Use of trans-signaling approach in chimeric antigen receptors

ABSTRACT

The present invention provides compositions and methods for inducing a CAR mediated trans-signal in a T cell. The trans-signaling CAR T cells comprise a first CAR having a first signaling module and a second CAR having a distinct second signaling module. The present invention also provides cells comprising a plurality of types of CARs, wherein the plurality of types of CARs participate in trans-signaling to induce T cell activation.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 14/044,569, filed Oct. 2, 2013, issued as U.S. Pat. No.10,117,896, which is entitled to priority under 35 U.S.C. § 119(e) toU.S. Provisional Patent Application No. 61/710,518, filed Oct. 5, 2012,each of which application is hereby incorporated by reference in theirentireties herein.

BACKGROUND OF THE INVENTION

The genetic redirection of T cells with chimeric antigen receptors(CARs) that link an antigen-specific single-chain antibody fragment(scFv) to intracellular signaling domains is at the forefront of cancerimmunotherapy (Jena et al., 2010, Blood 116:1035-1044; Gross et al.,1989, Proc. Natl. Acad. Sci. USA 86:10024-10028; Porter et al., 2011, N.Engl. J. Med. 365:725-733). CARs can functionally redirect T cells withhigh specificity to various surface antigens on tumor cells independentof MHC restriction and antigen processing, and therefore bypass majormechanisms by which tumors escape immune recognition. T cells bearing afirst generation CAR having only the T cell CD3z intracellular signalingdomain either fail to persist or become anergic since tumor cellsfrequently lack appropriate ligands for costimulatory molecules (Inmanet al., 2007, Curr. Cancer Drug Targets 7:15-30). Indeed, incompleteactivation of CART cells in vivo appears to limit their expansion andpersistence in vivo, and thus hampered their efficacy in clinical trialsin subjects with lymphoma (Till et al., 2008, Blood 112:2261-2271),neuroblastoma (Pule et al., 2008, Nat. Med. 14:1264-1270), or ovarian(Kershaw et al., 2006, Clin. Cancer Res. 12:6106-6115) or renal cancer(Lamers et al., 2006, J. Clin. Oncol. 24:e20-22).

To overcome these limitations, second generation CART cells weredeveloped that incorporate the intracellular domain of variouscostimulatory molecules such as CD28, 4-1BB, OX-40, and CD27, leading tosuperior expansion, persistence and activity of the CART cells inpreclinical mouse models (Carpenito et al., 2009, Proc. Natl. Acad. Sci.USA 106:3360-3365; Song et al., 2012, Blood 119:696-706) and in clinicalstudies (Porter et al., 2011, N. Engl. J. Med. 365:725-733; Kalos etal., 2011, Sci. Transl. Med. 3:95ra73; Savoldo et al., 2011, J. Clin.Invest. 121:1822-1826). However, the enhanced potency of the CARs can beassociated with autoimmunity due to on-target toxicities against normaltissues expressing the lower levels of the tumor associated antigens,with two serious adverse events (SAEs) reported so far.

The first SAE occurred in a clinical trial where administration ofanti-ErbB2 CART cells containing the CD28 and 4-1BB costimulatorysignaling regions into a patient with refractory colon cancer withmetastatic sites in lung and liver led to development of dramaticpulmonary toxicity with lung infiltrates and a cytokine storm followedby cardiac arrest and patient death (Morgan et al., 2010, Cancer J.16:336-341). In the second report, a lymphodepleted patient with bulkychronic lymphocytic leukemia received autologous T cells engineered withan anti-CD19 second-generation CAR containing the CD28 domain at a totaldose of 3×10⁷ T cells/kg. This patient developed fever, hypotension, anddyspnea 20 hours after infusion, which rapidly progressed. Low gradesepsis was the most likely reason of the patient's death, however thepossibility that a cyclophosphamide-induced “cytokine storm” may haveenhanced the in vivo activation of modified T cells is well considered(Brentjens et al., 2010, Mol. Ther. 18:666-668). It is therefore clearthat the development of strategies limiting potential early or latetoxicity is mandatory.

A fully human anti-mesothelin CAR capable of conferring potent in vitroand in vivo effector functions to primary T cells againstmesothelin-expressing tumors has previously been generated (Lanitis etal., 2012, Mol. Ther. 20:633-643). However, mesothelin CART cells holdthe potential to inflict damage against normal mesothelial cells liningthe pleura, peritoneum and peritoneum as well as epithelial cells of thetrachea, tonsils, fallopian tube and the rete testis which express lowlevels of mesothelin (Chang et al., 1992, Int. J. Cancer 50:373-381;Ordonez, 2003, Mod. Pathol. 16:192-197).

There is thus a need in the art for CAR therapies that do not exhibitserious adverse events. The present invention addresses this unmet needin the art.

BRIEF SUMMARY OF THE INVENTION

The invention includes a trans-signaling composition comprising anucleic acid molecule comprising a sequence encoding a first CAR and asecond CAR, wherein the first CAR comprises a first antigen bindingdomain, a first transmembrane domain, and an intracellular domain of acostimulatory molecule and wherein the second CAR comprises a secondantigen binding domain, a second transmembrane domain, and anintracellular domain of a T cell receptor.

In one embodiment, the costimulatory molecule is selected from the groupconsisting of CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, lymphocytefunction-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, aligand that specifically binds with CD83, and any combination thereof.In another embodiment, the intracellular domain of the T cell receptoris a CD3z signaling domain. In yet another embodiment, at least one ofthe first and second antigen binding domain is an antibody orantigen-binding fragment thereof. In a further embodiment,antigen-binding fragment is a Fab or a scFv. In another embodiment, thefirst and second antigen binding domains are different from one anotherand in a further embodiment, the first and second transmembrane domainsare different from one another. In an additional embodiment, the firstantigen binding domain binds a first target and the second antigenbinding domain binds a second target, wherein the first and secondtarget are each a tumor antigen associated with a solid tumor. Inanother embodiment, the tumor antigen is selected from the groupconsisting of folate (FRa), mesothelin, EGFRvIII, IL-13Ra, EGFR, CA-IX,MUC1, HER2, and any combination thereof. In yet a further embodiment,first antigen binding domain binds to a target selected from the groupconsisting of mesothelin and folate receptor, and wherein the secondantigen binding domain binds to a target selected from the groupconsisting of mesothelin and folate receptor.

The invention also includes a cell comprising a first CAR and a secondCAR, wherein the first CAR comprises a first antigen binding domain, afirst transmembrane domain, and an intracellular domain of acostimulatory molecule and wherein the second CAR comprises a secondantigen binding domain, a second transmembrane domain, and anintracellular domain of a T cell receptor. In one embodiment, the cellis a T cell. In another embodiment, the T cell is dependent on thebinding of the first CAR to its corresponding target and the binding ofthe second CAR to its corresponding target. In a further embodiment, thecell exhibits anti-tumor immunity when the first CAR binds to itscorresponding target and the second CAR binds to its correspondingtarget. In an additional embodiment, the cell exhibits heightened tumorspecificity.

Also included in the invention is a method for stimulating a Tcell-mediated immune response to a target cell population or tissue in amammal. The method comprises administering to a mammal an effectiveamount of a cell comprising a first CAR and a second CAR, wherein thefirst CAR comprises a first antigen binding domain, a firsttransmembrane domain, and an intracellular domain of a costimulatorymolecule and wherein the second CAR comprises a second antigen bindingdomain, a second transmembrane domain, and an intracellular domain of aT cell receptor, thereby stimulating a T cell-mediated immune responseto a target cell population or tissue in the mammal.

The invention further includes a method of providing an anti-tumorimmunity in a mammal. The method comprises administering to the mammalan effective amount of a cell comprising a first CAR and a second CAR,wherein the first CAR comprises a first antigen binding domain, a firsttransmembrane domain, and an intracellular domain of a costimulatorymolecule and wherein the second CAR comprises a second antigen bindingdomain, a second transmembrane domain, and an intracellular domain of aT cell receptor, thereby providing an anti-tumor immunity in the mammal.

Also included in the invention is a method of treating a mammal having adisease, disorder or condition associated with an elevated expression ofa tumor antigen. The method comprises administering to the mammal aneffective amount of a cell comprising a first CAR and a second CAR,wherein the first CAR comprises a first antigen binding domain, a firsttransmembrane domain, and an intracellular domain of a costimulatorymolecule and wherein the second CAR comprises a second antigen bindingdomain, a second transmembrane domain, and an intracellular domain of aT cell receptor, thereby treating the mammal.

The invention additionally includes a method of treating a human withcancer. The method comprises administering to the human a T cellcomprising a first CAR and a second CAR, wherein the first CAR comprisesa first antigen binding domain, a first transmembrane domain, and anintracellular domain of a costimulatory molecule and wherein the secondCAR comprises a second antigen binding domain, a second transmembranedomain, and an intracellular domain of a T cell receptor.

The invention further includes a method for enhancing a T cell-mediatedimmune response in a mammal. The method comprises administering aneffective amount of a cell genetically modified to express a CARcomprising an antigen binding domain, a transmembrane domain, and anintracellular domain of a costimulatory molecule, wherein CAR does notcomprise an intracellular domain of a T cell receptor.

In one embodiment, the costimulatory molecule is selected from the groupconsisting of CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, lymphocytefunction-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, aligand that specifically binds with CD83, and any combination thereof.In another embodiment, the antigen binding domain of the CAR is anantibody or antigen-binding fragment thereof.

In a further embodiment, the fragment is a Fab or a scFv. In yet anotherembodiment, the CAR functions with an independent receptor to induce a Tcell response. In an additional embodiment, the independent receptor isselected from the group consisting of an endogenous T cell receptor, anexogenous T cell receptor, a CD3z-containing CAR, and any combinationthereof.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description of preferred embodiments of theinvention will be better understood when read in conjunction with theappended drawings. For the purpose of illustrating the invention, thereare shown in the drawings embodiments which are presently preferred. Itshould be understood, however, that the invention is not limited to theprecise arrangements and instrumentalities of the embodiments shown inthe drawings.

FIG. 1, comprising FIGS. 1A-1C, depicts the generation and expression ofmesothelin and aFR specific CARs. FIG. 1A is a schematic representationof the anti-mesothelin (P4) based Chimeric Antigen Receptor (CAR)constructs containing the CD3ζ cytosolic domain alone (P4-z) or incombination with the CD28 costimulatory module (P4-28z). FIG. 1B is aschematic representation of the anti-aFR (F) based CAR constructscontaining the scFv (MOV-19) fused through the CD8 hinge and CD28 TM tothe CD28 costimulatory module (P4-28z). A truncated anti-aFR with nosignaling domains is shown. P4, anti-mesothelin scFv; VL, variable Lchain; L, Linker; VH, variable H chain; TM, transmembrane region. FIG.1C is a series of graphs depicting P4 CAR expression. P4 CAR expressionwas detected on human CD3-gated cells via recombinant mesothelin proteinstaining after transduction with lentivirus compared to untransduced Tcells. T cells bearing the F-28 CAR were detected via GFP transgeneexpression. Transduction efficiencies are indicated with the percentageof CAR expression of the transduced populations.

FIG. 2, comprising FIGS. 2A-2E, depicts the results of experimentsdemonstrating that trans-signaling CART cells exert superior antigenspecific cytokine secretion in vitro compared with first generation CART cells. FIG. 2A is a series of graphs depicting the surface expressionof mesothelin and/or FR by genetically modified C30 tumor cells. Thenative human ovarian cancer cell line C30 which does not express humanmesothelin or FRa was engineered to express high surface levels of humanmesothelin and/or FRa as shown by flow cytometry; Cells were alsostained with proper isotype antibody controls. FIG. 2B is a graphdepicting how primary human T cells transduced with P4-z and F-28 CARsexert superior IFN-g secretion compared to P4-z CAR T cells. TransducedT cells (1×10⁵ T cells) were cultured alone (none) or stimulatedovernight with an equal number of antigen-negative C30 cells or C30cells expressing mesothelin and/or FRa. Cell-free supernatant washarvested after about 20 hours of incubation and the IFN-g levels weremeasure with ELISA. FIG. 2C is a series of graphs depicting theco-expression of mesothelin and FRa in A1847 ovarian cancer cell line.FIG. 2D is a graph depicting how trans-signaling CART cells secretedhigher levels of IFN-γ in response to A1847. Transduced T cells (1×10⁵ Tcells) were cultured alone (none) or stimulated overnight with an equalnumber of antigen-negative C30 cells or A1857 cells expressingmesothelin and FRa. Cell-free supernatant was harvested after about 20hours of incubation and the IFN-γ levels were measure with ELISA. MeanIFN-γ concentration±SEM (pg/ml) from duplicate cultures is shown. FIG.2E is a graph depicting how trans-signaling CART cells secreted higherlevels of IL-2 in response to A1847. Cell-free supernatant from threeindependent cultures was harvested and pooled after 20 hours ofincubation and the IL-2 cytokine was quantified using cytometric beadarray technology. Values represent cytokine concentration (pg/ml)

FIG. 3, comprising FIGS. 3A-3B, depicts the results of experimentsdemonstrating that trans-signaling CAR T cells show equivalent in vitrocytolytic potency. FIG. 3A is a series of graphs depicting how dual CART cells exert superior degranulation and express T cell activationmarkers in response to mesothelin-specific stimulation. Firstgeneration, cis or trans costimulated CAR T cells were cultured with theindicated antigen-negative tumor cells or tumor cells expressing FRa ormesothelin or both antigens for 5 hours while being stained by ananti-CD107a, b antibody conjugated with FITC. After the incubationperiod, T cells were stained for CD8 and CD69 and analyzed by flowcytometry. FIG. 3B is a series of graphs depicting cytolytic function ofdual CAR transduced T cells. Primary human T cells transduced to expressM-z, M-z/F-28, M-28z, F-28 or GFP were co-cultured with Cr⁵¹-labelednative C30-F, C30-M and A1847 cancer cell lines for 4 hours at theindicated effector to target ratio. Percent specific target cell lysiswas calculated as (experimental−spontaneousrelease)÷(maximal−spontaneous release)×100. Error bars indicate standarddeviation.

FIG. 4, comprising FIGS. 4A-4B, depicts the results of experimentsdemonstrating that the integration of CD28 signaling in cis or in transincreases in vitro T-cell survival upon antigen specific stimulation andinhibition of antigen-induced post activation cell death. FIG. 4A is aseries of graphs depicting a mixed CAR T cell population containing dualCAR transduced T cells (M-z/F-28), single CAR transduced T cells (M-z orF-28) and untransduced T cells stimulated with A1847(M⁺/F⁺) cancer cellsfor three days. In parallel second generation M-28z CAR T cells wereco-cultured with A1847 for the same time period. After 3 days ofcoculture, apoptosis was quantified using annexin V and 7-AAD staining.FIG. 4B is a series of graphs depicting the absence of antigen inducedcell death (AICD) in CAR T cells stimulated with antigen negative cancercells. All the above CAR T cells populations were co-cultured with C30(M⁻/F⁻) for 3 days. Annexin V and 7-AAD staining was performed toevaluate apoptosis.

FIG. 5, comprising FIGS. 5A-5C, depicts the results of experimentsdemonstrating that trans signaling CAR T cells exert superior anti-tumoreffector functions in vivo compared with first generation CAR T cells.FIG. 5A is a graph depicting the in vivo suppression of largepre-established tumors by M-z/F-28 CAR T cells: effect of the CD28costimulatory signaling domain in trans. NSG mice bearing establisheds.c. tumor were treated with two i.v. injections of 7.5×10⁶ M-z,M-z/F-28 and M-28z CAR⁺ T cells or control anti-CD1928z and GFP T cellsor saline on 55 and 59 post-tumor inoculation. Tumor growth was assessedby caliper measurement. Tumors treated with M-z/F-28 and M-28zCAR-transduced T cells rapidly regressed (arrows indicate days of T cellinfusion); tumors treated with saline, GFP or CD19-28z CAR transduced Tcells did not regress 3 weeks post-first T cell dose (p<0.05). Equaldoses of P4-z CAR-transduced T cells only slowed the tumor growth(p=0.05). Results are expressed as a mean tumor volume (mm³±SEM) withn=5 for all groups. FIG. 5B is a graph depicting how the A1847 fLuc+bioluminescence signal is similar in M-z/F-28 and M-28z CAR treated miceand weaker than M-z treated mice 4 weeks after the first T cell dose.Control treatment groups show no decrement in the bioluminescencesignal. FIG. 5C is a series of photographs depicting the stablepersistence of CD28 cis or trans costimulated CAR engineered human Tcells in vivo. Peripheral blood was collected 2 weeks after the first Tcell infusion and quantified for the absolute number of human CD4⁺ andCD8⁺ T cells/ul of blood. Mean cell count±SEM is shown with n=5 for allgroups.

FIG. 6, comprising FIGS. 6A-6D, depicts the results of experimentsdemonstrating that trans-, but not cis-, signaling CAR T cells exhibitmore limited in vivo activity against cells bearing single antigen.FIGS. 6A-6C depict the in vivo anti-tumor efficacy of trans- orcis-signaling T cells against A1847 cells expressing or lacking FRa.5×10⁶ A1847(M⁺/F⁺) and A1847(M⁺/F⁻) cells were inoculated s.c.separately in the same NSG mice on opposite hind flanks. Mice with thetwo established A1847 (≥330mm³) tumors received tail vein injections ofCART cells on days 45 and 49 post-tumor inoculation. Tumor growth wasassessed by caliper measurement. FIG. 6A is a graph depicting howcontrol of A1847M⁺/F⁺ tumor outgrowth was identical between the transM-z/F-28 CART and cis-signaling M-28z CART cell groups. FIG. 6B is agraph depicting how inhibition of A1847M⁺/F⁻ outgrowth was partially butsignificantly attenuated in the trans M-z/F-28 CART cell group comparedwith the cis M-28z mice group. Results are expressed as a mean tumorvolume (mm³±SEM) with n=5 for all groups. FIG. 6C is a graph depictinghow trans-signaling CART cells were statistically less effective ininhibiting the outgrowth of A1847M⁺/F⁻, compared with their activityagainst the A1847M⁺/F⁺ tumor in the same mice. Results are expressed asa mean tumor volume (mm³±SEM) with n=5 for all groups. FIG. 6D is agraph depicting how the FLuc+ bioluminescence signal from A1847M⁺/F⁺tumors is dramatically lower compared with the signal derived from theA1847M⁺/F⁻ tumors in the same mice treated with M-z/F-28 CAR T cells 4weeks after the first T cell dose. FLuc+ bioluminescence signal from thedifferent types of tumor is low and similar in the M-28z treated mousegroup. Control treatment groups show no decrement in the bioluminescencesignal.

FIG. 7, comprising FIGS. 7A-7B, depicts the results of experimentsdemonstrating that trans signaling CAR T-cell preferentially localize todual antigen-expressing tumors in vivo. FIG. 7A is a series ofphotographs depicting NSG mice with s.c. established A1847 tumorsexpressing or not expressing FRa in opposite flanks. The mice weretreated with two i.v. injections of 7.5×10⁶ T cells expressing CD19-28z(top), M-z/F-28 (middle), or M-28z (bottom) on days 45 and 49 post-tumorinoculation. Mice were euthanized after four weeks and tumors werecollected and stained for human CD3 expression (brown) of proper isotypecontrol. Representative sections for both tumor derivatives are shown at×10 magnifications. FIG. 7B is a graph depicting the quantification ofCD3⁺ T cells within the A1847 (M+/F+) and A1847 (M+/F−) tumors in thedifferentially treated groups. CD3⁺ T cells were counted in 10 randomlyselected intratumoral fields of each slide at high magnification (20×).Data are expressed as the means±SE with n=5 for all groups.

FIG. 8 is an image depicting the correlation of GFP transgene expressionand Protein L binding to the scFv. F-28 CAR T cells were lentivirallytransduced and 5 days after were stained with biotinylated Protein-Lfollowed by SA-APC. Transduction efficiency was also monitored using GFPtransgene expression. Transduction efficiencies are indicated with thepercentage of CAR expression of the transduced populations.

FIG. 9, comprising FIGS. 9A-9B, depicts the results of experimentsdemonstrating that trans-, but not cis-, signaling CAR T cells exhibitmore limited in vitro activity against cells bearing single antigen.FIG. 9A is a series of graphs depicting the surface expression ofmesothelin and/or FR by genetically modified A1847 tumor cells. Thenative human ovarian cancer cell line A1847 which express humanmesothelin and FRa was transduced with lentiviral particles encoding foran shRNA specific for silencing aFR gene expression. Cells were alsostained with an anti-mesothelin reagent (P4 Bb) or proper isotypeantibody controls. aFR expression was unaltered after engineering cellswith control shRNA (A1847M+/F+). FIG. 9B is a graph depicting IFN-γsecretion by trans M-z/F-28 CART cells was significantly reduced inresponse to A1847M+/F− compared with A1847M+/F+. Transduced T cells(1×10⁵ T cells) were cultured alone (none) or stimulated overnight withan equal number of C30 cells, (A1847M+/F+) or (A1847M+/F−). Cell-freesupernatant was harvested after about 20 hours of incubation and theIFN-γ levels were measure with ELISA. Mean IFN-γ concentration±SEM(pg/ml) from duplicate cultures is shown.

FIG. 10 is a schematic illustration depicting the dissociation ofsignaling molecules in two distinct CARs.

FIG. 11, comprising FIGS. 11A-11C, depicts a schematic illustration ofthe costimulatory domain in trans-signaling CAR T cells bearing 4-1BBexerting superior IFN-g secretion compared with the P4-z CAR T cellsagainst meso/folate expressing tumor cells. FIG. 11A is a schematicrepresentation of the first generation CAR and the second generationCAR, which contains CD3z and CD28 in a cis-signaling conformation. FIG.11B depicts a costimulatory CAR comprising the 4-1BB intracellularsignaling domain. FIG. 11C is a graph depicting the functionality of theFR-BB costimulatory CAR in the trans-signaling approach.

DETAILED DESCRIPTION

The present invention provides compositions and methods to regulate thespecificity and activity of T cells modified to express a chimericantigen receptor (CAR). T cells that have been genetically modified toexpress a CAR have been used in treatments for cancers where the CARredirects the modified T cell to recognize a tumor antigen. The presentinvention also provides cells comprising a plurality of types of CARs,wherein the plurality of types of CARs participate in trans-signaling toinduce T cell activation. In this aspect, CAR as used herein refers to achimeric protein that comprises an antigen binding moiety and anintracellular signaling moiety. In some instances, it may be beneficialto effectively control and regulate CAR T cells such that they killtumor cells while not affecting normal bystander cells. Thus, in oneembodiment, the present invention also provides methods of killingcancerous cells while minimizing the depletion of normal non-cancerouscells, thereby improving the specificity of CAR therapy.

The present invention also provides a trans-signaling CAR approach. Thetrans-signaling CAR approach includes the physical separation of aplurality of types of CARs expressed on a cell, where binding of aplurality of types of CARs to their target antigen is required for CAR Tcell activation. For example in the trans-signaling CAR approach, eachCAR from the plurality of type of CARs have different intracellularsignaling domain. For example, when a plurality of types of CARs is usedto induce CAR T cell activation, the first type of CAR can only comprisean intracellular domain from a T cell receptor and the second type ofCAR can only comprise an intracellular domain from a co-stimulatorymolecule. In this manner, optimal CAR T cell activation occurs only whenthe intracellular domain of the T cell receptor from the first CAR isactive and the intracellular domain of the co-stimulatory molecule fromthe second CAR is active in the T cell.

In one embodiment, the methods of the invention comprise geneticallymodifying a T cell to express a plurality of types of CARs, where T cellactivation is dependent on the binding of a plurality of types of CARsto their target antigens. In one embodiment, dependence on the bindingof a plurality of different CARs improves the specificity of CAR T celltherapies. In one embodiment, the trans-signaling CAR cells comprise afirst signal module and a distinct second signal module which areincorporated into two distinct CARs, a first CAR and a second CAR,respectively, each with a different antigen specificity. In oneembodiment, activation of the modified T cell only occurs when the firstCAR binds the first desired antigen and the second CAR binds to thesecond desired antigen. In one embodiment, the first CAR comprises aprimary signaling domain derived from CD3z, and the second CAR comprisesa costimulatory signaling region.

In one embodiment, the genetically modified trans-signaling CAR T cellsof the invention can be generated by introducing a lentiviral vectorcomprising one or more desired CARs into a T cell. In one embodiment,the modified T cells of the invention can be generated by introducing aplurality of lentiviral vectors each comprising one or more desired CARsinto a T cell. In one embodiment, the modified T cells of the inventionare able to replicate in vivo resulting in long-term persistence thatcan lead to sustained tumor control.

In one embodiment, the genetically modified trans-signaling CAR T cellsof the invention can be generated by transfecting an RNA encoding one ormore desired CARs into a T cell. In one embodiment, the modified T cellsof the invention can be generated by transfecting a plurality of RNAsequences each encoding one ore more desired CARs into a T cell. In oneembodiment, both the CAR and the bispecific antibody are transientlyexpressed in the genetically modified T cells.

In one embodiment, the genetically modified trans-signaling CAR T cellsof the invention can be generated by transfecting an RNA encoding one ormore desired CARs into a T cell, and introducing a lentiviral vectorcomprising one or more desired CARs into the T cell.

The present invention also provides costimulatory-only CARs.Costimulatory-only CARs comprise at least one costimulatory signalingregion. In one embodiment, costimulatory-only CARs augment T cellactivation through signaling mediated by the costimulatory signalingregion. In one embodiment, binding of the costimulatory-only CAR to itstarget antigen is insufficient to induce T cell activation on its own.For example, the costimulatory-only CAR of the invention functions incombination with at least one other independent receptor of the T cellto induce T cell activation. The other independent receptor includes,but is not limited to, an endogenous T cell receptor, an exogenous Tcell receptor, a CD3z containing CAR, and the like. The presentinvention also provides methods of improving T cell activation byadministering a costimulatory-only CAR to a T cell or T cell population.In one embodiment, the T cell is used in adoptive T cell therapy oradoptive T cell transfer.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention pertains. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice for testing of the present invention, the preferredmaterials and methods are described herein. In describing and claimingthe present invention, the following terminology will be used.

It is also to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting.

The articles “a” and “an” are used herein to refer to one or toplurality (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or pluralityelement.

“About” as used herein when referring to a measurable value such as anamount, a temporal duration, and the like, is meant to encompassvariations of ±20% or ±10%, more preferably ±5%, even more preferably±1%, and still more preferably ±0.1% from the specified value, as suchvariations are appropriate to perform the disclosed methods.

“Activation,” as used herein, refers to the state of a T cell that hasbeen sufficiently stimulated to induce detectable cellularproliferation. Activation can also be associated with induced cytokineproduction, and detectable effector functions. The term “activated Tcells” refers to, among other things, T cells that are undergoing celldivision.

The term “antibody,” as used herein, refers to an immunoglobulinmolecule which specifically binds with an antigen. Antibodies can beintact immunoglobulins derived from natural sources or from recombinantsources and can be immunoreactive portions of intact immunoglobulins.Antibodies are often of immunoglobulin molecules. The antibodies in thepresent invention may exist in a variety of forms including, forexample, polyclonal antibodies, monoclonal antibodies, Fv, Fab andF(ab)2, as well as single chain antibodies and humanized antibodies(Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratory Press, NY; Harlow et al., 1989, In: Antibodies:A Laboratory Manual, Cold Spring Harbor, N.Y.; Houston et al., 1988,Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science242:423-426).

The term “antibody fragment” refers to a portion of an intact antibodyand refers to the antigenic determining variable regions of an intactantibody. Examples of antibody fragments include, but are not limitedto, Fab, Fab′, F(ab′)2, and Fv fragments, linear antibodies, scFvantibodies, and multispecific antibodies formed from antibody fragments.

An “antibody heavy chain,” as used herein, refers to the larger of thetwo types of polypeptide chains present in all antibody molecules intheir naturally occurring conformations.

An “antibody light chain,” as used herein, refers to the smaller of thetwo types of polypeptide chains present in all antibody molecules intheir naturally occurring conformations. □ and □ light chains refer tothe two major antibody light chain isotypes.

By the term “synthetic antibody” as used herein, is meant an antibodywhich is generated using recombinant DNA technology, such as, forexample, an antibody expressed by a bacteriophage as described herein.The term should also be construed to mean an antibody which has beengenerated by the synthesis of a DNA molecule encoding the antibody andwhich DNA molecule expresses an antibody protein, or an amino acidsequence specifying the antibody, wherein the DNA or amino acid sequencehas been obtained using synthetic DNA or amino acid sequence technologywhich is available and well known in the art.

The term “antigen” or “Ag” as used herein is defined as a molecule thatprovokes an immune response. This immune response may involve eitherantibody production, or the activation of specificimmunologically-competent cells, or both. The skilled artisan willunderstand that any macromolecule, including virtually all proteins orpeptides, can serve as an antigen. Furthermore, antigens can be derivedfrom recombinant or genomic DNA. A skilled artisan will understand thatany DNA, which comprises a nucleotide sequences or a partial nucleotidesequence encoding a protein that elicits an immune response thereforeencodes an “antigen” as that term is used herein. Furthermore, oneskilled in the art will understand that an antigen need not be encodedsolely by a full length nucleotide sequence of a gene. It is readilyapparent that the present invention includes, but is not limited to, theuse of partial nucleotide sequences of one, or more than one, gene andthat these nucleotide sequences are arranged in various combinations toelicit the desired immune response. Moreover, a skilled artisan willunderstand that an antigen need not be encoded by a “gene” at all. It isreadily apparent that an antigen can be generated synthesized or can bederived from a biological sample. Such a biological sample can include,but is not limited to a tissue sample, a tumor sample, a cell or abiological fluid.

The term “anti-tumor effect” as used herein, refers to a biologicaleffect which can be manifested by a decrease in tumor volume, a decreasein the number of tumor cells, a decrease in the number of metastases, anincrease in life expectancy, or amelioration of various physiologicalsymptoms associated with the cancerous condition. An “anti-tumor effect”can also be manifested by the ability of the peptides, polynucleotides,cells and antibodies of the invention in prevention of the occurrence oftumor in the first place.

The term “auto-antigen” means, in accordance with the present invention,any self-antigen which is recognized by the immune system as if it wereforeign. Auto-antigens comprise, but are not limited to, cellularproteins, phosphoproteins, cellular surface proteins, cellular lipids,nucleic acids, glycoproteins, including cell surface receptors.

The term “autoimmune disease” as used herein is defined as a disorderthat results from an autoimmune response. An autoimmune disease is theresult of an inappropriate and excessive response to a self-antigen.Examples of autoimmune diseases include but are not limited to,Addision's disease, alopecia areata, ankylosing spondylitis, autoimmunehepatitis, autoimmune parotitis, Crohn's disease, diabetes (Type I),dystrophic epidermolysis bullosa, epididymitis, glomerulonephritis,Graves' disease, Guillain-Barr syndrome, Hashimoto's disease, hemolyticanemia, systemic lupus erythematosus, multiple sclerosis, myastheniagravis, pemphigus vulgaris, psoriasis, rheumatic fever, rheumatoidarthritis, sarcoidosis, scleroderma, Sjogren's syndrome,spondyloarthropathies, thyroiditis, vasculitis, vitiligo, myxedema,pernicious anemia, ulcerative colitis, among others.

As used herein, the term “autologous” is meant to refer to any materialderived from the same individual to which it is later to bere-introduced into the individual.

“Allogeneic” refers to a graft derived from a different animal of thesame species.

“Xenogeneic” refers to a graft derived from an animal of a differentspecies.

The term “cancer” as used herein is defined as disease characterized bythe rapid and uncontrolled growth of aberrant cells. Cancer cells canspread locally or through the bloodstream and lymphatic system to otherparts of the body. Examples of various cancers include but are notlimited to, breast cancer, prostate cancer, ovarian cancer, cervicalcancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer,liver cancer, brain cancer, lymphoma, leukemia, lung cancer and thelike.

“Co-stimulatory ligand,” as the term is used herein, includes a moleculeon an antigen presenting cell (e.g., an aAPC, dendritic cell, B cell,and the like) that specifically binds a cognate co-stimulatory moleculeon a T cell, thereby providing a signal which, in addition to theprimary signal provided by, for instance, binding of a TCR/CD3 complexwith an MHC molecule loaded with peptide, mediates a T cell response,including, but not limited to, proliferation, activation,differentiation, and the like. A co-stimulatory ligand can include, butis not limited to, CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL,OX40L, inducible costimulatory ligand (ICOS-L), intercellular adhesionmolecule (ICAM), CD3OL, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM,lymphotoxin beta receptor, 3/TR6, ILT3, ILT4, HVEM, an agonist orantibody that binds Toll ligand receptor and a ligand that specificallybinds with B7-H3. A co-stimulatory ligand also encompasses, inter alia,an antibody that specifically binds with a co-stimulatory moleculepresent on a T cell, such as, but not limited to, CD27, CD28, 4-1BB,OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1(LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specificallybinds with CD83.

A “co-stimulatory molecule” refers to the cognate binding partner on a Tcell that specifically binds with a co-stimulatory ligand, therebymediating a co-stimulatory response by the T cell, such as, but notlimited to, proliferation. Co-stimulatory molecules include, but are notlimited to an MHC class I molecule, BTLA and a Toll ligand receptor.

A “co-stimulatory signal”, as used herein, refers to a signal, which incombination with a primary signal, such as TCR/CD3 ligation, leads to Tcell proliferation and/or upregulation or downregulation of keymolecules.

A “disease” is a state of health of an animal wherein the animal cannotmaintain homeostasis, and wherein if the disease is not ameliorated thenthe animal's health continues to deteriorate. In contrast, a “disorder”in an animal is a state of health in which the animal is able tomaintain homeostasis, but in which the animal's state of health is lessfavorable than it would be in the absence of the disorder. Leftuntreated, a disorder does not necessarily cause a further decrease inthe animal's state of health.

An “effective amount” as used herein, means an amount which provides atherapeutic or prophylactic benefit.

“Encoding” refers to the inherent property of specific sequences ofnucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, toserve as templates for synthesis of other polymers and macromolecules inbiological processes having either a defined sequence of nucleotides(i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and thebiological properties resulting therefrom. Thus, a gene encodes aprotein if transcription and translation of mRNA corresponding to thatgene produces the protein in a cell or other biological system. Both thecoding strand, the nucleotide sequence of which is identical to the mRNAsequence and is usually provided in sequence listings, and thenon-coding strand, used as the template for transcription of a gene orcDNA, can be referred to as encoding the protein or other product ofthat gene or cDNA.

As used herein “endogenous” refers to any material from or producedinside an organism, cell, tissue or system.

As used herein, the term “exogenous” refers to any material introducedto an organism, cell, tissue or system that was produced outside anorganism, cell, tissue or system.

The term “expression” as used herein is defined as the transcriptionand/or translation of a particular nucleotide sequence.

“Expression vector” refers to a vector comprising a recombinantpolynucleotide comprising expression control sequences operativelylinked to a nucleotide sequence to be expressed. An expression vectorcomprises sufficient cis-acting elements for expression; other elementsfor expression can be supplied by the host cell or in an in vitroexpression system. Expression vectors include all those known in theart, such as cosmids, plasmids (e.g., naked or contained in liposomes)and viruses (e.g., lentiviruses, retroviruses, adenoviruses, andadeno-associated viruses) that incorporate the recombinantpolynucleotide.

“Homologous” refers to the sequence similarity or sequence identitybetween two polypeptides or between two nucleic acid molecules. When aposition in both of the two compared sequences is occupied by the samebase or amino acid monomer subunit, e.g., if a position in each of twoDNA molecules is occupied by adenine, then the molecules are homologousat that position. The percent of homology between two sequences is afunction of the number of matching or homologous positions shared by thetwo sequences divided by the number of positions compared X 100. Forexample, if 6 of 10 of the positions in two sequences are matched orhomologous then the two sequences are 60% homologous. By way of example,the DNA sequences ATTGCC and TATGGC share 50% homology. Generally, acomparison is made when two sequences are aligned to give maximumhomology.

The term “immunoglobulin” or “Ig,” as used herein is defined as a classof proteins, which function as antibodies. Antibodies expressed by Bcells are sometimes referred to as the BCR (B cell receptor) or antigenreceptor. The five members included in this class of proteins are IgA,IgG, IgM, IgD, and IgE. IgA is the primary antibody that is present inbody secretions, such as saliva, tears, breast milk, gastrointestinalsecretions and mucus secretions of the respiratory and genitourinarytracts. IgG is the most common circulating antibody. IgM is the mainimmunoglobulin produced in the primary immune response in most subjects.It is the most efficient immunoglobulin in agglutination, complementfixation, and other antibody responses, and is important in defenseagainst bacteria and viruses. IgD is the immunoglobulin that has noknown antibody function, but may serve as an antigen receptor. IgE isthe immunoglobulin that mediates immediate hypersensitivity by causingrelease of mediators from mast cells and basophils upon exposure toallergen.

As used herein, an “instructional material” includes a publication, arecording, a diagram, or any other medium of expression which can beused to communicate the usefulness of the compositions and methods ofthe invention. The instructional material of the kit of the inventionmay, for example, be affixed to a container which contains the nucleicacid, peptide, and/or composition of the invention or be shippedtogether with a container which contains the nucleic acid, peptide,and/or composition. Alternatively, the instructional material may beshipped separately from the container with the intention that theinstructional material and the compound be used cooperatively by therecipient.

“Isolated” means altered or removed from the natural state. For example,a nucleic acid or a peptide naturally present in a living animal is not“isolated,” but the same nucleic acid or peptide partially or completelyseparated from the coexisting materials of its natural state is“isolated.” An isolated nucleic acid or protein can exist insubstantially purified form, or can exist in a non-native environmentsuch as, for example, a host cell.

In the context of the present invention, the following abbreviations forthe commonly occurring nucleic acid bases are used. “A” refers toadenosine, “C” refers to cytosine, “G” refers to guanosine, “T” refersto thymidine, and “U” refers to uridine.

Unless otherwise specified, a “nucleotide sequence encoding an aminoacid sequence” includes all nucleotide sequences that are degenerateversions of each other and that encode the same amino acid sequence. Thephrase nucleotide sequence that encodes a protein or an RNA may alsoinclude introns to the extent that the nucleotide sequence encoding theprotein may in some version contain an intron(s).

A “lentivirus” as used herein refers to a genus of the Retroviridaefamily. Lentiviruses are unique among the retroviruses in being able toinfect non-dividing cells; they can deliver a significant amount ofgenetic information into the DNA of the host cell, so they are one ofthe most efficient methods of a gene delivery vector. HIV, SIV, and FIVare all examples of lentiviruses. Vectors derived from lentivirusesoffer the means to achieve significant levels of gene transfer in vivo.

By the term “modulating,” as used herein, is meant mediating adetectable increase or decrease in the level of a response in a subjectcompared with the level of a response in the subject in the absence of atreatment or compound, and/or compared with the level of a response inan otherwise identical but untreated subject. The term encompassesperturbing and/or affecting a native signal or response therebymediating a beneficial therapeutic response in a subject, preferably, ahuman.

Unless otherwise specified, a “nucleotide sequence encoding an aminoacid sequence” includes all nucleotide sequences that are degenerateversions of each other and that encode the same amino acid sequence.Nucleotide sequences that encode proteins and RNA may include introns.

The term “operably linked” refers to functional linkage between aregulatory sequence and a heterologous nucleic acid sequence resultingin expression of the latter. For example, a first nucleic acid sequenceis operably linked with a second nucleic acid sequence when the firstnucleic acid sequence is placed in a functional relationship with thesecond nucleic acid sequence. For instance, a promoter is operablylinked to a coding sequence if the promoter affects the transcription orexpression of the coding sequence. Generally, operably linked DNAsequences are contiguous and, where necessary to join two protein codingregions, in the same reading frame.

The term “overexpressed” tumor antigen or “overexpression” of a tumorantigen is intended to indicate an abnormal level of expression of atumor antigen in a cell from a disease area like a solid tumor within aspecific tissue or organ of the patient relative to the level ofexpression in a normal cell from that tissue or organ. Patients havingsolid tumors or a hematological malignancy characterized byoverexpression of the tumor antigen can be determined by standard assaysknown in the art.

“Parenteral” administration of an immunogenic composition includes,e.g., subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), orintrasternal injection, or infusion techniques.

The terms “patient,” “subject,” “individual,” and the like are usedinterchangeably herein, and refer to any animal, or cells thereofwhether in vitro or in situ, amenable to the methods described herein.In certain non-limiting embodiments, the patient, subject or individualis a human.

The term “polynucleotide” as used herein is defined as a chain ofnucleotides. Furthermore, nucleic acids are polymers of nucleotides.Thus, nucleic acids and polynucleotides as used herein areinterchangeable. One skilled in the art has the general knowledge thatnucleic acids are polynucleotides, which can be hydrolyzed into themonomeric “nucleotides.” The monomeric nucleotides can be hydrolyzedinto nucleosides. As used herein polynucleotides include, but are notlimited to, all nucleic acid sequences which are obtained by any meansavailable in the art, including, without limitation, recombinant means,i.e., the cloning of nucleic acid sequences from a recombinant libraryor a cell genome, using ordinary cloning technology and PCR, and thelike, and by synthetic means.

As used herein, the terms “peptide,” “polypeptide,” and “protein” areused interchangeably, and refer to a compound comprised of amino acidresidues covalently linked by peptide bonds. A protein or peptide mustcontain at least two amino acids, and no limitation is placed on themaximum number of amino acids that can comprise a protein's or peptide'ssequence. Polypeptides include any peptide or protein comprising two ormore amino acids joined to each other by peptide bonds. As used herein,the term refers to both short chains, which also commonly are referredto in the art as peptides, oligopeptides and oligomers, for example, andto longer chains, which generally are referred to in the art asproteins, of which there are many types. “Polypeptides” include, forexample, biologically active fragments, substantially homologouspolypeptides, oligopeptides, homodimers, heterodimers, variants ofpolypeptides, modified polypeptides, derivatives, analogs, fusionproteins, among others. The polypeptides include natural peptides,recombinant peptides, synthetic peptides, or a combination thereof.

The term “promoter” as used herein is defined as a DNA sequencerecognized by the synthetic machinery of the cell, or introducedsynthetic machinery, required to initiate the specific transcription ofa polynucleotide sequence.

As used herein, the term “promoter/regulatory sequence” means a nucleicacid sequence which is required for expression of a gene productoperably linked to the promoter/regulatory sequence. In some instances,this sequence may be the core promoter sequence and in other instances,this sequence may also include an enhancer sequence and other regulatoryelements which are required for expression of the gene product. Thepromoter/regulatory sequence may, for example, be one which expressesthe gene product in a tissue specific manner.

A “constitutive” promoter is a nucleotide sequence which, when operablylinked with a polynucleotide which encodes or specifies a gene product,causes the gene product to be produced in a cell under most or allphysiological conditions of the cell.

An “inducible” promoter is a nucleotide sequence which, when operablylinked with a polynucleotide which encodes or specifies a gene product,causes the gene product to be produced in a cell substantially only whenan inducer which corresponds to the promoter is present in the cell.

A “tissue-specific” promoter is a nucleotide sequence which, whenoperably linked with a polynucleotide encodes or specified by a gene,causes the gene product to be produced in a cell substantially only ifthe cell is a cell of the tissue type corresponding to the promoter.

By the term “specifically binds,” as used herein with respect to anantibody, is meant an antibody which recognizes a specific antigen, butdoes not substantially recognize or bind other molecules in a sample.For example, an antibody that specifically binds to an antigen from onespecies may also bind to that antigen from one or more species. But,such cross-species reactivity does not itself alter the classificationof an antibody as specific. In another example, an antibody thatspecifically binds to an antigen may also bind to different allelicforms of the antigen. However, such cross reactivity does not itselfalter the classification of an antibody as specific. In some instances,the terms “specific binding” or “specifically binding,” can be used inreference to the interaction of an antibody, a protein, or a peptidewith a second chemical species, to mean that the interaction isdependent upon the presence of a particular structure (e.g., anantigenic determinant or epitope) on the chemical species; for example,an antibody recognizes and binds to a specific protein structure ratherthan to proteins generally. If an antibody is specific for epitope “A,”the presence of a molecule containing epitope A (or free, unlabeled A),in a reaction containing labeled “A” and the antibody, will reduce theamount of labeled A bound to the antibody.

By the term “stimulation,” is meant a primary response induced bybinding of a stimulatory molecule (e.g., a TCR/CD3 complex) with itscognate ligand thereby mediating a signal transduction event, such as,but not limited to, signal transduction via the TCR/CD3 complex.Stimulation can mediate altered expression of certain molecules, such asdownregulation of TGF-β, and/or reorganization of cytoskeletalstructures, and the like.

A “stimulatory molecule,” as the term is used herein, means a moleculeon a T cell that specifically binds with a cognate stimulatory ligandpresent on an antigen presenting cell.

A “stimulatory ligand,” as used herein, means a ligand that when presenton an antigen presenting cell (e.g., an aAPC, a dendritic cell, aB-cell, and the like) can specifically bind with a cognate bindingpartner (referred to herein as a “stimulatory molecule”) on a T cell,thereby mediating a primary response by the T cell, including, but notlimited to, activation, initiation of an immune response, proliferation,and the like. Stimulatory ligands are well-known in the art andencompass, inter alia, an WIC Class I molecule loaded with a peptide, ananti-CD3 antibody, a superagonist anti-CD28 antibody, and a superagonistanti-CD2 antibody.

The term “subject,” “patient” and “individual” are used interchangeablyherein and are intended to include living organisms in which an immuneresponse can be elicited (e.g., mammals). Examples of subjects includehumans, dogs, cats, mice, rats, and transgenic species thereof.

As used herein, a “substantially purified” cell is a cell that isessentially free of other cell types. A substantially purified cell alsorefers to a cell which has been separated from other cell types withwhich it is normally associated in its naturally occurring state. Insome instances, a population of substantially purified cells refers to ahomogenous population of cells. In other instances, this term referssimply to cell that have been separated from the cells with which theyare naturally associated in their natural state. In some embodiments,the cells are cultured in vitro. In other embodiments, the cells are notcultured in vitro.

The term “therapeutic” as used herein means a treatment and/orprophylaxis. A therapeutic effect is obtained by suppression, remission,or eradication of a disease state.

The term “therapeutically effective amount” refers to the amount of thesubject compound that will elicit the biological or medical response ofa tissue, system, or subject that is being sought by the researcher,veterinarian, medical doctor or other clinician. The term“therapeutically effective amount” includes that amount of a compoundthat, when administered, is sufficient to prevent development of, oralleviate to some extent, one or more of the signs or symptoms of thedisorder or disease being treated. The therapeutically effective amountwill vary depending on the compound, the disease and its severity andthe age, weight, etc., of the subject to be treated.

To “treat” a disease as the term is used herein, means to reduce thefrequency or severity of at least one sign or symptom of a disease ordisorder experienced by a subject.

The term “transfected” or “transformed” or “transduced” as used hereinrefers to a process by which exogenous nucleic acid is transferred to,or introduced into, the host cell. A “transfected” or “transformed” or“transduced” cell is one which has been transfected, transformed ortransduced with exogenous nucleic acid. The cell includes the primarysubject cell and its progeny.

The phrase “under transcriptional control” or “operatively linked” asused herein means that the promoter is in the correct location andorientation in relation to a polynucleotide to control the initiation oftranscription by RNA polymerase and expression of the polynucleotide.

A “vector” is a composition of matter which comprises an isolatednucleic acid and which can be used to deliver the isolated nucleic acidto the interior of a cell. Numerous vectors are known in the artincluding, but not limited to, linear polynucleotides, polynucleotidesassociated with ionic or amphiphilic compounds, plasmids, and viruses.Thus, the term “vector” includes an autonomously replicating plasmid ora virus. The term should also be construed to include non-plasmid andnon-viral compounds which facilitate transfer of nucleic acid intocells, such as, for example, polylysine compounds, liposomes, and thelike. Examples of viral vectors include, but are not limited to,adenoviral vectors, adeno-associated virus vectors, retroviral vectors,and the like.

Ranges: throughout this disclosure, various aspects of the invention canbe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. Thisapplies regardless of the breadth of the range.

Description

The present invention provides compositions and methods fortrans-signaling CAR cells, where binding of a plurality of types of CARsto their target antigen is required for CAR T cell activation.Dependence on the binding of a plurality of types of CARs improves thespecificity of the lytic activity of the CAR T cell, thereby reducingthe potential for depleting normal healthy tissue. In one embodiment,the trans-signaling CAR cells of the present invention comprise a firstsignal module and a second signal module which are incorporated into twodistinct CARs, a first CAR and a second CAR, respectively, each withdifferent antigen specificity and signaling modules.

In one embodiment, the first type of CAR only comprises an intracellulardomain from a T cell receptor and the second type of CAR only comprisesan intracellular domain from a co-stimulatory molecule. In this manner,optimal CAR T cell activation occurs when the intracellular domain ofthe T cell receptor from the first CAR is activated and theintracellular domain of the co-stimulatory molecule from the second CARis activated in the T cell.

In one embodiment, the first CAR comprises a CD3z signaling domain andthe second CAR comprises a costimulatory signaling region. This isbecause the present invention is partly based on the discovery that atrans-signaling CAR-mediated T-cell response comprising a CAR of each ofa CD3z signaling and a costimulatory signaling region produces improvedcytokine secretion and resistance to antigen-induced cell death (AICD)in vitro. In one embodiment, the trans-signaling CAR cells provideincreased IL-2 production. In one embodiment, the trans-signaling CARcells of the invention exhibit superior tumor specificity compared tocells expressing a single type of CAR comprising a costimulatory domainand a CD3z signaling domain. In one embodiment, the invention comprisesCAR cells with a first CAR comprising a CD3z signaling domain, and asecond CAR comprising a CD28 costimulatory signaling region.

The trans-signaling CAR cells of the present invention are comprised ofa plurality of types of CARs, each specific for a different antigen. Inone embodiment, a first CAR targets folate receptor (aFR), while asecond CAR targets mesothelin (meso). This is because the presentinvention is partly based on the discovery that the CAR cells comprisingthe dual specificity can be selective for tumor over normal tissuesexpressing the lower levels of the tumor associated antigens (TAA),thereby reducing the potential for “on-target” toxicity, whilemaintaining potent anti-cancer activity, tumor localization andpersistence in vivo as compared to first generation CARs. In oneembodiment, T cell activation and lytic activity of trans-signaling CARcells requires the co-expression of both aFR and mesothelin on the tumorcell surface. For example, ovarian cancers are known to express both aFRand mesothelin. As a result, trans-signaling CAR cells showed reducedtargeting of cells expressing only one tumor associated antigen ascompared to conventional second generation CARs, where CD3z andcostimulatory signaling regions are in tandem. In one embodiment, theTAAs are nearly uniformly expressed on the surface of a cell of aparticular cancer, while the TAAs are expressed in low andnon-overlapping levels in normal tissue.

In some embodiments, the present invention is directed to retroviral orlentiviral vectors encoding one or more CARs stably integrated into a Tcell and stably expressed therein. In other embodiments, the presentinvention is directed to RNA encoding one or more CARs that istransfected into a T cell and transiently expressed therein. Transient,non-integrated expression of the CARs mitigates concerns associated withpermanent and integrated expression in a cell.

In one embodiment, the present invention provides methods to treatcancer and other disorders using CAR T cell therapy while limiting thedepletion of healthy bystander cells. In one embodiment, theaccumulation of T cells in tumors is increased when both antigens areexpressed on the tumor cell surface as compared to only one antigen. Inone embodiment, the dual CAR T cell approach can modestly improve safetyfor clinical application without significantly diminishing its antitumorpotential. Additional approaches to limiting CART mediated autoimmuneeffects are also considered. In one embodiment, the optimal T cell dosecan be defined by a careful design of a dose-escalation strategy. In oneembodiment, conditional suicide genes can be co-expressed to overcomepotential side effects of non-tumor cell recognition by CAR T cells. Forexample, HSV-TK, i-Casp9, the cytoplasmic domain of Fas, or an induciblecaspase can be incorporated into genetically engineered T cells toprevent aberrant T cell responses. In one embodiment, T cells can beelectroporated with optimized RNAs encoding for CARs to allow fortransient CAR expression.

The antigen specificity of each CAR is not limited by whether the CARcomprises a signaling domain or a costimulatory signaling region. In oneembodiment, the first CAR comprises a CD3z signaling domain and amesothelin binding domain, and the second CAR comprises a CD28costimulatory signaling region and a folate receptor binding domain. Inone embodiment, the first CAR comprises a CD3z signaling domain and afolate receptor binding domain, and the second CAR comprises a CD28costimulatory signaling region and a mesothelin binding domain. Asdescribed elsewhere herein, T cells modified to express a plurality oftypes of CARs can be generated by administering lentiviral vectors, invitro transcribed RNA, or combination thereof, to the cells. Thetrans-signaling CAR cells can therefore be engineered to target anycombination of antigens on the surface of a particular cell of interestin order to enhance the binding affinity of the CAR cells toward thecell of interest as well as to reduce non-selective binding to normaltissues.

The present invention also provides costimulatory-only CARs. In oneembodiment, the costimulatory-only CAR comprises an antigen bindingdomain and at least one costimulatory domain. For example, in oneembodiment, the costimulatory-only CAR of the invention comprises a CD28costimulatory signaling region. In one embodiment, thecostimulatory-only CAR of the invention comprises a 4-1BB (CD137)costimulatory signaling domain. In one embodiment, binding of thecostimulatory-only CAR to its target antigen is insufficient to inducesubstantial T cell activation. In one embodiment, the costimulatory-onlydomain lacks a CD3z domain. In one embodiment, costimulatory-only CARsare used to enhance endogenous T cell activation. In another embodiment,costimulatory-only CARs are used to enhance T cell activation in T cellsused for adoptive T cell transfer.

In one embodiment, the invention comprises controlling or regulating CART cell activity. In one embodiment, the invention comprises compositionsand methods related to genetically modifying T cells to express aplurality of types of CARs, where CAR T cell activation is dependent onthe binding of a plurality of types of CARs to their target receptor.

In one embodiment, the present invention provides methods for treatingcancer and other disorders using CAR T cell therapies while minimizingthe depletion of normal healthy tissue. The cancer may be ahematological malignancy, a solid tumor, a primary or a metastasizingtumor. Other diseases treatable using the compositions and methods ofthe invention include viral, bacterial and parasitic infections as wellas autoimmune diseases.

Chimeric Antigen Receptors

The present invention provides a chimeric antigen receptor (CAR)comprising an extracellular and intracellular domain. Compositions andmethods of making CARs have been described in PCT/US11/64191, which isincorporated in its entirety by reference herein.

The extracellular domain comprises a target-specific binding elementotherwise referred to as an antigen binding domain. In some embodiments,the extracellular domain also comprises a hinge domain. In oneembodiment, the hinge domain is CD8α. In one embodiment, theintracellular domain or otherwise the cytoplasmic domain comprises azeta chain portion. In one embodiment, the intracellular domain orotherwise the cytoplasmic domain comprises a costimulatory signalingregion. The costimulatory signaling region refers to a portion of theCAR comprising the intracellular domain of a costimulatory molecule.Costimulatory molecules are cell surface molecules other than antigenreceptors or their ligands that are required for an efficient responseof lymphocytes to antigen.

Between the extracellular domain and the transmembrane domain of theCAR, or between the cytoplasmic domain and the transmembrane domain ofthe CAR, there may be incorporated a spacer domain. As used herein, theterm “spacer domain” generally means any oligo- or polypeptide thatfunctions to link the transmembrane domain to, either the extracellulardomain or, the cytoplasmic domain in the polypeptide chain. A spacerdomain may comprise up to 300 amino acids, preferably 10 to 100 aminoacids and most preferably 25 to 50 amino acids.

The present invention includes retroviral and lentiviral vectorconstructs expressing a CAR that can be directly transduced into a cell.The present invention also includes an RNA construct that can bedirectly transfected into a cell. A method for generating mRNA for usein transfection involves in vitro transcription (IVT) of a template withspecially designed primers, followed by polyA addition, to produce aconstruct containing 3′ and 5′ untranslated sequence (“UTR”), a 5′ capand/or Internal Ribosome Entry Site (IRES), the gene to be expressed,and a polyA tail, typically 50-2000 bases in length. RNA so produced canefficiently transfect different kinds of cells. In one embodiment, thetemplate includes sequences for the CAR.

Preferably, the CAR comprises an extracellular domain, a transmembranedomain and a cytoplasmic domain. The extracellular domain andtransmembrane domain can be derived from any desired source of suchdomains. In some instances, the hinge domain of the CAR of the inventioncomprises the CD8α hinge domain.

In one embodiment, the CAR of the invention comprises a target-specificbinding element otherwise referred to as an antigen binding domain. Thechoice of moiety depends upon the type and number of ligands that definethe surface of a target cell. For example, the antigen binding domainmay be chosen to recognize a ligand that acts as a cell surface markeron target cells associated with a particular disease state. Thusexamples of cell surface markers that may act as ligands for the antigenmoiety domain in the CAR of the invention include those associated withviral, bacterial and parasitic infections, autoimmune disease and cancercells.

In one embodiment, the CAR of the invention can be engineered to targeta tumor antigen of interest by way of engineering a desired antigenbinding domain that specifically binds to an antigen on a tumor cell. Inthe context of the present invention, “tumor antigen” or“hyperporoliferative disorder antigen” or “antigen associated with ahyperproliferative disorder,” refers to antigens that are common tospecific hyperproliferative disorders such as cancer. The antigensdiscussed herein are merely included by way of example. The list is notintended to be exclusive and further examples will be readily apparentto those of skill in the art.

Tumor antigens are proteins that are produced by tumor cells that elicitan immune response, particularly T-cell mediated immune responses. Theselection of the antigen binding domain of the invention will depend onthe particular type of cancer to be treated. Tumor antigens are wellknown in the art and include, for example, a glioma-associated antigen,carcinoembryonic antigen (CEA), β-human chorionic gonadotropin,alphafetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1,MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS),intestinal carboxyl esterase, mut hsp70-2, M-CSF, prostase,prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53, prostein,PSMA, Her2/neu, survivin and telomerase, prostate-carcinoma tumorantigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrinB2, CD22,insulin growth factor (IGF)-I, IGF-II, IGF-I receptor, folate receptor(FRa) and mesothelin. In a preferred embodiment, the tumor antigen isselected from the group consisting of folate receptor (FRa), mesothelin,EGFRvIII, IL-13Ra, EGFR, CA-IX, MUC1, HER2, and any combination thereof.In one embodiment, the first CAR comprises an antigen binding domainwhich binds to mesothelin and the second CAR comprises an antigenbinding domain that binds to FRa. In one embodiment, the CAR comprisesan antigen binding domain that binds to HER2.

In one embodiment, the tumor antigen comprises one or more antigeniccancer epitopes associated with a malignant tumor. Malignant tumorsexpress a number of proteins that can serve as target antigens for animmune attack. These molecules include but are not limited totissue-specific antigens such as MART-1, tyrosinase and GP 100 inmelanoma and prostatic acid phosphatase (PAP) and prostate-specificantigen (PSA) in prostate cancer. Other target molecules belong to thegroup of transformation-related molecules such as the oncogeneHER-2/Neu/ErbB-2. Yet another group of target antigens are onco-fetalantigens such as carcinoembryonic antigen (CEA). In B-cell lymphoma thetumor-specific idiotype immunoglobulin constitutes a trulytumor-specific immunoglobulin antigen that is unique to the individualtumor. B-cell differentiation antigens such as CD19, CD20 and CD37 areother candidates for target antigens in B-cell lymphoma. Some of theseantigens (CEA, HER-2, CD19, CD20, idiotype) have been used as targetsfor passive immunotherapy with monoclonal antibodies with limitedsuccess.

The type of tumor antigen referred to in the invention may also be atumor-specific antigen (TSA) or a tumor-associated antigen (TAA). A TSAis unique to tumor cells and does not occur on other cells in the body.A TAA associated antigen is not unique to a tumor cell and instead isalso expressed on a normal cell under conditions that fail to induce astate of immunologic tolerance to the antigen. The expression of theantigen on the tumor may occur under conditions that enable the immunesystem to respond to the antigen. TAAs may be antigens that areexpressed on normal cells during fetal development when the immunesystem is immature and unable to respond or they may be antigens thatare normally present at extremely low levels on normal cells but whichare expressed at much higher levels on tumor cells.

Non-limiting examples of TSA or TAA antigens include the following:Differentiation antigens such as MART-1/MelanA (MART-I), gp100 (Pmel17), tyrosinase, TRP-1, TRP-2 and tumor-specific multilineage antigenssuch as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15; overexpressedembryonic antigens such as CEA; overexpressed oncogenes and mutatedtumor-suppressor genes such as p53, Ras, HER-2/neu; unique tumorantigens resulting from chromosomal translocations; such as BCR-ABL,E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens, such as theEpstein Barr virus antigens EBVA and the human papillomavirus (HPV)antigens E6 and E7. Other large, protein-based antigens include TSP-180,MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, p185erbB2, p180erbB-3, c-met,nm-23H1, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras,beta-Catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72,alpha-fetoprotein, beta-HCG, BCA225, BTAA, CA 125, CA 15-3\CA27.29\BCAA, CA 195, CA 242, CA-50, CAM43, CD68\P1, CO-029, FGF-5, G250,Ga733\EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB/70K, NY-CO-1,RCAS1, SDCCAG16, TA-90\Mac-2 binding protein\cyclophilin C-associatedprotein, TAAL6, TAG72, TLP, and TPS.

In a preferred embodiment, the antigen binding domain portion of the CARtargets an antigen that includes but is not limited to CD19, CD20, CD22,ROR1, Mesothelin, CD33/IL3Ra, c-Met, PSMA, Glycolipid F77, EGFRvIII,GD-2, MY-ESO-1 TCR, MAGE A3 TCR, folate receptor (FRa), and the like.

Depending on the desired antigen to be targeted, the CAR of theinvention can be engineered to include the appropriate antigen bindmoiety that is specific to the desired antigen target.

The antigen binding domain can be any domain that binds to the antigenincluding but not limited to monoclonal antibodies, polyclonalantibodies, synthetic antibodies, human antibodies, humanizedantibodies, and fragments thereof. In some instances, it is beneficialfor the antigen binding domain to be derived from the same species inwhich the CAR will ultimately be used in. For example, for use inhumans, it may be beneficial for the antigen binding domain of the CARto comprise a human antibody or fragment thereof. Thus, in oneembodiment, the antigen biding domain portion comprises a human antibodyor a fragment thereof. Alternatively, in some embodiments, a non-humanantibody is humanized, where specific sequences or regions of theantibody are modified to increase similarity to an antibody naturallyproduced in a human.

In one embodiment of the present invention, a plurality of types of CARsis expressed on the surface of a T cell. The different types of CAR maydiffer in their antigen binding domain. That is, in one embodiment, thedifferent types of CARs each bind a different antigen. In oneembodiment, the different antigens are markers for a specific tumor. Forexample, in one embodiment, the different types of CARs each bind to adifferent antigen, where each antigen is expressed on a specific type oftumor. Examples of such antigens are discussed elsewhere herein.

With respect to the transmembrane domain, the CAR can be designed tocomprise a transmembrane domain that is fused to the extracellulardomain of the CAR. In one embodiment, the transmembrane domain thatnaturally is associated with one of the domains in the CAR is used. Insome instances, the transmembrane domain can be selected or modified byamino acid substitution to avoid binding of such domains to thetransmembrane domains of the same or different surface membrane proteinsto minimize interactions with other members of the receptor complex.

The transmembrane domain may be derived either from a natural or from asynthetic source. Where the source is natural, the domain may be derivedfrom any membrane-bound or transmembrane protein. Transmembrane regionsof particular use in this invention may be derived from (i.e. compriseat least the transmembrane region(s) of) the alpha, beta or zeta chainof the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9,CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, ICOS.Alternatively the transmembrane domain may be synthetic, in which caseit will comprise predominantly hydrophobic residues such as leucine andvaline. Preferably a triplet of phenylalanine, tryptophan and valinewill be found at each end of a synthetic transmembrane domain.Optionally, a short oligo- or polypeptide linker, preferably between 2and 10 amino acids in length may form the linkage between thetransmembrane domain and the cytoplasmic signaling domain of the CAR. Aglycine-serine doublet provides a particularly suitable linker.

The cytoplasmic domain or otherwise the intracellular signaling domainof the CAR of the invention influences the activation of at least one ofthe normal effector functions of the immune cell in which the CAR hasbeen placed in. The term “effector function” refers to a specializedfunction of a cell. Effector function of a T cell, for example, may becytolytic activity or helper activity including the secretion ofcytokines. Thus the term “intracellular signaling domain” refers to theportion of a protein which transduces the effector function signal anddirects the cell to perform a specialized function. While usually theentire intracellular signaling domain can be employed, in many cases itis not necessary to use the entire chain. To the extent that a truncatedportion of the intracellular signaling domain is used, such truncatedportion may be used in place of the intact chain as long as ittransduces the effector function signal. The term intracellularsignaling domain is thus meant to include any truncated portion of theintracellular signaling domain sufficient to transduce the effectorfunction signal.

In one embodiment of the present invention, the effector function of thecell is dependent upon the binding of a plurality of types of CARs totheir targeted antigen. For example, in one embodiment, binding of onetype of CAR to its target is not sufficient to induce the effectorfunction of the cell.

Examples of intracellular signaling domains for use in the CAR of theinvention include the cytoplasmic sequences of the T cell receptor (TCR)and co-receptors that act in concert to initiate signal transductionfollowing antigen receptor engagement, as well as any derivative orvariant of these sequences and any synthetic sequence that has the samefunctional capability.

It is known that signals generated through the TCR alone areinsufficient for full activation of the T cell and that a secondary orco-stimulatory signal is also required. Thus, T cell activation can besaid to be mediated by two distinct classes of cytoplasmic signalingsequence: those that initiate antigen-dependent primary activationthrough the TCR (primary cytoplasmic signaling sequences) and those thatact in an antigen-independent manner to provide a secondary orco-stimulatory signal (secondary cytoplasmic signaling sequences).

Primary cytoplasmic signaling sequences regulate primary activation ofthe TCR complex either in a stimulatory way, or in an inhibitory way.Primary cytoplasmic signaling sequences that act in a stimulatory mannermay contain signaling motifs which are known as immunoreceptortyrosine-based activation motifs or ITAMs.

Examples of ITAM containing primary cytoplasmic signaling sequences thatare of particular use in the invention include those derived from TCRzeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CDS, CD22,CD79a, CD79b, and CD66d. It is particularly preferred that cytoplasmicsignaling molecule in at least one CAR of the trans-signaling CAR cellsof the invention comprises a cytoplasmic signaling sequence derived fromCD3z.

In one embodiment, the cytoplasmic domain of the CAR can be designed tocomprise a costimulatory signaling region. The costimulatory signalingregion refers to a portion of the CAR comprising the intracellulardomain of a costimulatory molecule. A costimulatory molecule is a cellsurface molecule other than an antigen receptor or their ligands that isrequired for an efficient response of lymphocytes to an antigen.Examples of such molecules include CD27, CD28, 4-1BB (CD137), OX40,CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1(LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specificallybinds with CD83, and the like.

The cytoplasmic signaling sequences within the cytoplasmic signalingportion of the CAR of the invention may be linked to each other in arandom or specified order. Optionally, a short oligo- or polypeptidelinker, preferably between 2 and 10 amino acids in length may form thelinkage. A glycine-serine doublet provides a particularly suitablelinker.

In one embodiment, the cytoplasmic domain is designed to comprise thesignaling domain of CD3-zeta. In another embodiment, the cytoplasmicdomain is designed to comprise the signaling domain of 4-1BB. In anotherembodiment, the cytoplasmic domain is designed to comprise the signalingdomain of CD28. In one embodiment of the present invention, a pluralityof types of CARs is expressed on a cell, where the different types ofCAR may vary in their cytoplasmic domain. In one embodiment, at leastone type of CAR comprises the CD3z domain, while at least one type ofCAR comprises a costimulatory signaling region, for example the 4-1BBdomain. However, the different types of CARs are not limited by anyparticular cytoplasmic domain. For example, each type of CAR cancomprise any ITAM containing sequence, costimulatory signaling region,or combination thereof such that binding of each individual type of CARis insufficient to induce effector function but binding of a pluralityof types of CARs are able to induce effector function. That is, thedomains of each type of CAR work together to induce effector function.

The present invention further provides for a costimulatory-only CAR,wherein the CAR comprises an antigen binding domain and at least onecostimulatory domain. In a preferred embodiment, the costimulatory-onlydomain lacks a primary signaling domain, such as CD3z or other primarysignaling domains detailed elsewhere herein. A costimulatory-only CARcomprises an extracellular domain, a transmembrane domain and acytoplasmic domain. The cytoplasmic domain comprises a costimulatorymolecule, such as those described elsewhere herein. In one embodiment,the costimulatory molecule is selected from the group consisting of CD28and 4-1BB (CD137). A costimulatory-only CAR cell is independent of a CARcomprising a primary signaling domain, such as CD3z or those describedelsewhere herein. In one embodiment, a costimulatory-only CAR can beused to enhance endogenous T cell activation.

A costimulatory-only CAR can function in T cell in combination with anysignal playing a role in T cell effector function, including, but notlimited to, signals from an endogenous TCR, and exogenous TCR providedby gene transfer, and a first generation CD3z-only CAR, the like. In oneembodiment, the costimulatory-only CAR comprises a CD28 costimulatorysignaling region. In one embodiment, the costimulatory-only CARcomprises a 4-1BB costimulatory signaling region.

In one embodiment, the costimulatory-only CAR is not capable of inducingsubstantial T cell activation upon binding of its target antigen. Forexample, recognition of aFR alone and singular transmission of CD28signal, in the absence of mesothelin-directed CD3z-signals, does notactivate trans-signaling CAR T cells. Instead, the costimulatory CAR isenhancing the signal induced by a CAR comprising a signaling domainderived from CD3z or those described elsewhere herein.

In one embodiment, the costimulatory-only CAR can be used in a method ofusing a plurality of different CARs to enhance tumor specificity. Forexample, in one embodiment, the activation of a T cell comprising afirst CAR comprising a primary signaling domain and a second CARcomprising a costimulatory domain requires binding to a tumor cell whichexpresses each antigens specific to each CAR. T cell activation would belimited against cells bearing only a single antigen, thereby permittingthe T cell response to be selective for the tumor cell. In oneembodiment, a costimulatory-only CAR can increase the resistance of CART cells to antigen-induced cell death (AICD). In one embodiment, acostimulatory-only CAR can provide sustained survival and activationagainst normal host tissues expressing low levels of TAA.

In one embodiment, a costimulatory-only CAR provides antigen-triggeredcostimulation to antigen-specific T cells where recognition/TCRsignaling occurs via an independent receptor. In one embodiment, theindependent receptor is a first generation CD3z-only CAR. In oneembodiment, the independent receptor is an endogenous TCR. In oneembodiment, the independent receptor is an exogenous transferred TCR. Inone embodiment, the costimulatory-only CAR comprises the nucleic acidsequence of one of SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10. In oneembodiment, the costimulatory-only CAR comprises the nucleic acidsequence that encodes the amino acid sequence of one of SEQ ID NO: 5,SEQ ID NO: 7, or SEQ ID NO: 9. In another embodiment, thecostimulatory-only CAR comprises the amino acid sequence of one of SEQID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9.

RNA Transfection

In one embodiment, the genetically modified T cells of the invention aremodified through the introduction of RNA. In one embodiment, an in vitrotranscribed RNA CAR can be introduced to a cell as a form of transienttransfection. In another embodiment, the RNA CAR is introduced alongwith an in vitro transcribed RNA encoding a bispecific antibody. The RNAis produced by in vitro transcription using a polymerase chain reaction(PCR)-generated template. DNA of interest from any source can bedirectly converted by PCR into a template for in vitro mRNA synthesisusing appropriate primers and RNA polymerase. The source of the DNA canbe, for example, genomic DNA, plasmid DNA, phage DNA, cDNA, syntheticDNA sequence or any other appropriate source of DNA. The desiredtemplate for in vitro transcription is the CAR of the present invention.In one embodiment, the template for the RNA CAR comprises anextracellular domain comprising a single chain variable domain of ananti-tumor antibody; a transmembrane domain comprising the hinge andtransmembrane domain of CD8a; and a cytoplasmic domain. By way ofanother example, the template comprises a plurality of types of CARs.

In one embodiment, the DNA to be used for PCR contains an open readingframe. The DNA can be from a naturally occurring DNA sequence from thegenome of an organism. In one embodiment, the DNA is a full length geneof interest of a portion of a gene. The gene can include some or all ofthe 5′ and/or 3′ untranslated regions (UTRs). The gene can include exonsand introns. In one embodiment, the DNA to be used for PCR is a humangene. In another embodiment, the DNA to be used for PCR is a human geneincluding the 5′ and 3′ UTRs. The DNA can alternatively be an artificialDNA sequence that is not normally expressed in a naturally occurringorganism. An exemplary artificial DNA sequence is one that containsportions of genes that are ligated together to form an open readingframe that encodes a fusion protein. The portions of DNA that areligated together can be from a single organism or from pluralityorganism.

Genes that can be used as sources of DNA for PCR include genes thatencode polypeptides that provide a therapeutic or prophylactic effect toan organism or that can be used to diagnose a disease or disorder in anorganism. Preferred genes are genes which are useful for a short termtreatment, or where there are safety concerns regarding dosage or theexpressed gene. For example, for treatment of cancer, autoimmunedisorders, parasitic, viral, bacterial, fungal or other infections, thetransgene(s) to be expressed may encode a polypeptide that functions asa ligand or receptor for cells of the immune system, or can function tostimulate or inhibit the immune system of an organism. In someembodiments, it is not desirable to have prolonged ongoing stimulationof the immune system, nor necessary to produce changes which last aftersuccessful treatment, since this may then elicit a new problem. Fortreatment of an autoimmune disorder, it may be desirable to inhibit orsuppress the immune system during a flare-up, but not long term, whichcould result in the patient becoming overly sensitive to an infection.

PCR is used to generate a template for in vitro transcription of mRNAwhich is used for transfection. Methods for performing PCR are wellknown in the art. Primers for use in PCR are designed to have regionsthat are substantially complementary to regions of the DNA to be used asa template for the PCR. “Substantially complementary”, as used herein,refers to sequences of nucleotides where a majority or all of the basesin the primer sequence are complementary, or one or more bases arenon-complementary, or mismatched. Substantially complementary sequencesare able to anneal or hybridize with the intended DNA target underannealing conditions used for PCR. The primers can be designed to besubstantially complementary to any portion of the DNA template. Forexample, the primers can be designed to amplify the portion of a genethat is normally transcribed in cells (the open reading frame),including 5′ and 3′ UTRs. The primers can also be designed to amplify aportion of a gene that encodes a particular domain of interest. In oneembodiment, the primers are designed to amplify the coding region of ahuman cDNA, including all or portions of the 5′ and 3′ UTRs. Primersuseful for PCR are generated by synthetic methods that are well known inthe art. “Forward primers” are primers that contain a region ofnucleotides that are substantially complementary to nucleotides on theDNA template that are upstream of the DNA sequence that is to beamplified. “Upstream” is used herein to refer to a location 5, to theDNA sequence to be amplified relative to the coding strand. “Reverseprimers” are primers that contain a region of nucleotides that aresubstantially complementary to a double-stranded DNA template that aredownstream of the DNA sequence that is to be amplified. “Downstream” isused herein to refer to a location 3′ to the DNA sequence to beamplified relative to the coding strand.

Any DNA polymerase useful for PCR can be used in the methods disclosedherein. The reagents and polymerase are commercially available from anumber of sources.

Chemical structures with the ability to promote stability and/ortranslation efficiency may also be used. The RNA preferably has 5′ and3′ UTRs. In one embodiment, the 5′ UTR is between zero and 3000nucleotides in length. The length of 5′ and 3′ UTR sequences to be addedto the coding region can be altered by different methods, including, butnot limited to, designing primers for PCR that anneal to differentregions of the UTRs. Using this approach, one of ordinary skill in theart can modify the 5′ and 3′ UTR lengths required to achieve optimaltranslation efficiency following transfection of the transcribed RNA.

The 5′ and 3′ UTRs can be the naturally occurring, endogenous 5′ and 3′UTRs for the gene of interest. Alternatively, UTR sequences that are notendogenous to the gene of interest can be added by incorporating the UTRsequences into the forward and reverse primers or by any othermodifications of the template. The use of UTR sequences that are notendogenous to the gene of interest can be useful for modifying thestability and/or translation efficiency of the RNA. For example, it isknown that AU-rich elements in 3′ UTR sequences can decrease thestability of mRNA. Therefore, 3′ UTRs can be selected or designed toincrease the stability of the transcribed RNA based on properties ofUTRs that are well known in the art.

In one embodiment, the 5′ UTR can contain the Kozak sequence of theendogenous gene. Alternatively, when a 5′ UTR that is not endogenous tothe gene of interest is being added by PCR as described above, aconsensus Kozak sequence can be redesigned by adding the 5′ UTRsequence. Kozak sequences can increase the efficiency of translation ofsome RNA transcripts, but does not appear to be required for all RNAs toenable efficient translation. The requirement for Kozak sequences formany mRNAs is known in the art. In other embodiments the 5′ UTR can bederived from an RNA virus whose RNA genome is stable in cells. In otherembodiments various nucleotide analogues can be used in the 3′ or 5′ UTRto impede exonuclease degradation of the mRNA.

To enable synthesis of RNA from a DNA template without the need for genecloning, a promoter of transcription should be attached to the DNAtemplate upstream of the sequence to be transcribed. When a sequencethat functions as a promoter for an RNA polymerase is added to the 5′end of the forward primer, the RNA polymerase promoter becomesincorporated into the PCR product upstream of the open reading framethat is to be transcribed. In one preferred embodiment, the promoter isa T7 polymerase promoter, as described elsewhere herein. Other usefulpromoters include, but are not limited to, T3 and SP6 RNA polymerasepromoters. Consensus nucleotide sequences for T7, T3 and SP6 promotersare known in the art.

In a preferred embodiment, the mRNA has both a cap on the 5′ end and a3′ poly(A) tail which determine ribosome binding, initiation oftranslation and stability mRNA in the cell. On a circular DNA template,for instance, plasmid DNA, RNA polymerase produces a long concatamericproduct which is not suitable for expression in eukaryotic cells. Thetranscription of plasmid DNA linearized at the end of the 3′ UTR resultsin normal sized mRNA which is not effective in eukaryotic transfectioneven if it is polyadenylated after transcription.

On a linear DNA template, phage T7 RNA polymerase can extend the 3′ endof the transcript beyond the last base of the template (Schenborn andMierendorf, Nuc Acids Res., 13:6223-36 (1985); Nacheva andBerzal-Herranz, Eur. J. Biochem., 270:1485-65 (2003).

The conventional method of integration of polyA/T stretches into a DNAtemplate is molecular cloning. However polyA/T sequence integrated intoplasmid DNA can cause plasmid instability, which is why plasmid DNAtemplates obtained from bacterial cells are often highly contaminatedwith deletions and other aberrations. This makes cloning procedures notonly laborious and time consuming but often not reliable. That is why amethod which allows construction of DNA templates with polyA/T 3′stretch without cloning highly desirable.

The polyA/T segment of the transcriptional DNA template can be producedduring PCR by using a reverse primer containing a polyT tail, such as100T tail (size can be 50-5000 T), or after PCR by any other method,including, but not limited to, DNA ligation or in vitro recombination.Poly(A) tails also provide stability to RNAs and reduce theirdegradation. Generally, the length of a poly(A) tail positivelycorrelates with the stability of the transcribed RNA. In one embodiment,the poly(A) tail is between 100 and 5000 adenosines.

Poly(A) tails of RNAs can be further extended following in vitrotranscription with the use of a poly(A) polymerase, such as E. colipolyA polymerase (E-PAP). In one embodiment, increasing the length of apoly(A) tail from 100 nucleotides to between 300 and 400 nucleotidesresults in about a two-fold increase in the translation efficiency ofthe RNA. Additionally, the attachment of different chemical groups tothe 3′ end can increase mRNA stability. Such attachment can containmodified/artificial nucleotides, aptamers and other compounds. Forexample, ATP analogs can be incorporated into the poly(A) tail usingpoly(A) polymerase. ATP analogs can further increase the stability ofthe RNA.

5′ caps on also provide stability to RNA molecules. In a preferredembodiment, RNAs produced by the methods disclosed herein include a 5′cap. The 5′ cap is provided using techniques known in the art anddescribed herein (Cougot, et al., Trends in Biochem. Sci., 29:436-444(2001); Stepinski, et al., RNA, 7:1468-95 (2001); Elango, et al.,Biochim. Biophys. Res. Commun., 330:958-966 (2005)).

The RNAs produced by the methods disclosed herein can also contain aninternal ribosome entry site (IRES) sequence. The IRES sequence may beany viral, chromosomal or artificially designed sequence which initiatescap-independent ribosome binding to mRNA and facilitates the initiationof translation. Any solutes suitable for cell electroporation, which cancontain factors facilitating cellular permeability and viability such assugars, peptides, lipids, proteins, antioxidants, and surfactants can beincluded.

RNA can be introduced into target cells using any of a number ofdifferent methods, for instance, commercially available methods whichinclude, but are not limited to, electroporation (Amaxa Nucleofector-II(Amaxa Biosystems, Cologne, Germany)), (ECM 830 (BTX) (HarvardInstruments, Boston, Mass.) or the Gene Pulser II (BioRad, Denver,Colo.), Multiporator (Eppendort, Hamburg Germany), cationic liposornemediated transfection using lipofection, polymer encapsulation, peptidemediated transfection, or biolistic particle delivery systems such as“gene guns” (see, for example, Nishikawa, et al. Hum Gene Ther.,12(8):861-70 (2001).

Genetically Modified T Cells

In some embodiments, the CAR sequences are delivered into cells using aretroviral or lentiviral vector. CAR-expressing retroviral andlentiviral vectors can be delivered into different types of eukaryoticcells as well as into tissues and whole organisms using transduced cellsas carriers or cell-free local or systemic delivery of encapsulated,bound or naked vectors. The method used can be for any purpose wherestable expression is required or sufficient.

In other embodiments, the CAR sequences are delivered into cells usingin vitro transcribed mRNA. In vitro transcribed mRNA CAR can bedelivered into different types of eukaryotic cells as well as intotissues and whole organisms using transfected cells as carriers orcell-free local or systemic delivery of encapsulated, bound or nakedmRNA. The method used can be for any purpose where transient expressionis required or sufficient.

In one embodiment, the genetically modified cells express one or moretypes of CARs, where the nucleic acid sequence of each type of CAR isset forth in SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, and SEQ ID NO:10. In one embodiment, the genetically modified cells express one ormore types of CARs, where the nucleic acid sequence of the types of CARsencode the amino acid sequences set forth in SEQ ID NO: 3, SEQ ID NO: 5,SEQ ID NO: 7, and SEQ ID NO: 9. In another embodiment, the geneticallymodified cells express one or more types of CARs, where the amino acidsequence of the types of CARs is set forth in SEQ ID NO: 3, SEQ ID NO:5, SEQ ID NO: 7, and SEQ ID NO: 9.

The disclosed methods can be applied to the modulation of T cellactivity in basic research and therapy, in the fields of cancer, stemcells, acute and chronic infections, and autoimmune diseases, includingthe assessment of the ability of the genetically modified T cell to killa target cancer cell.

The methods also provide the ability to control the level of expressionover a wide range by changing, for example, the promoter or the amountof input RNA, making it possible to individually regulate the expressionlevel. Furthermore, the PCR-based technique of mRNA production greatlyfacilitates the design of the chimeric receptor mRNAs with differentstructures and combination of their domains. For example, varying ofdifferent intracellular effector/costimulator domains on multiplechimeric receptors in the same cell allows determination of thestructure of the receptor combinations which assess the highest level ofcytotoxicity against multi-antigenic targets, and at the same timelowest cytotoxicity toward normal cells.

One advantage of RNA transfection methods of the invention is that RNAtransfection is essentially transient and a vector-free: An RNAtransgene can be delivered to a lymphocyte and expressed thereinfollowing a brief in vitro cell activation, as a minimal expressingcassette without the need for any additional viral sequences. Underthese conditions, integration of the transgene into the host cell genomeis unlikely. Cloning of cells is not necessary because of the efficiencyof transfection of the RNA and its ability to uniformly modify theentire lymphocyte population.

Genetic modification of T cells with in vitro-transcribed RNA (IVT-RNA)makes use of two different strategies both of which have beensuccessively tested in various animal models. Cells are transfected within vitro-transcribed RNA by means of lipofection or electroporation.Preferably, it is desirable to stabilize IVT-RNA using variousmodifications in order to achieve prolonged expression of transferredIVT-RNA.

Some IVT vectors are known in the literature which are utilized in astandardized manner as template for in vitro transcription and whichhave been genetically modified in such a way that stabilized RNAtranscripts are produced. Currently protocols used in the art are basedon a plasmid vector with the following structure: a 5′ RNA polymerasepromoter enabling RNA transcription, followed by a gene of interestwhich is flanked either 3′ and/or 5′ by untranslated regions (UTR), anda 3′ polyadenyl cassette containing 50-70 A nucleotides. Prior to invitro transcription, the circular plasmid is linearized downstream ofthe polyadenyl cassette by type II restriction enzymes (recognitionsequence corresponds to cleavage site). The polyadenyl cassette thuscorresponds to the later poly(A) sequence in the transcript. As a resultof this procedure, some nucleotides remain as part of the enzymecleavage site after linearization and extend or mask the poly(A)sequence at the 3′ end. It is not clear, whether this nonphysiologicaloverhang affects the amount of protein produced intracellularly fromsuch a construct.

RNA has several advantages over more traditional plasmid or viralapproaches. Gene expression from an RNA source does not requiretranscription and the protein product is produced rapidly after thetransfection. Further, since the RNA has to only gain access to thecytoplasm, rather than the nucleus, and therefore typical transfectionmethods result in an extremely high rate of transfection. In addition,plasmid based approaches require that the promoter driving theexpression of the gene of interest be active in the cells under study.

In another aspect, the RNA construct can be delivered into the cells byelectroporation. See, e.g., the formulations and methodology ofelectroporation of nucleic acid constructs into mammalian cells astaught in US 2004/0014645, US 2005/0052630A1, US 2005/0070841A1, US2004/0059285A1, US 2004/0092907A1. The various parameters includingelectric field strength required for electroporation of any known celltype are generally known in the relevant research literature as well asnumerous patents and applications in the field. See e.g., U.S. Pat. Nos.6,678,556, 7,171,264, and 7,173,116. Apparatus for therapeuticapplication of electroporation are available commercially, e.g., theMedPulser™ DNA Electroporation Therapy System (Inovio/Genetronics, SanDiego, Calif.), and are described in patents such as U.S. Pat. Nos.6,567,694; 6,516,223, 5,993,434, 6,181,964, 6,241,701, and 6,233,482;electroporation may also be used for transfection of cells in vitro asdescribed e.g. in US20070128708A1. Electroporation may also be utilizedto deliver nucleic acids into cells in vitro. Accordingly,electroporation-mediated administration into cells of nucleic acidsincluding expression constructs utilizing any of the many availabledevices and electroporation systems known to those of skill in the artpresents an exciting new means for delivering an RNA of interest to atarget cell.

Activation and Expansion of T Cells

Whether prior to or after genetic modification of the T cells to expressa desirable CAR, the T cells can be activated and expanded generallyusing methods as described, for example, in U.S. Pat. Nos. 6,352,694;6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681;7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223;6,905,874; 6,797,514; 6,867,041; and U.S. Patent Application PublicationNo. 20060121005.

Generally, the T cells of the invention are expanded by contact with asurface having attached thereto an agent that stimulates a CD3/TCRcomplex associated signal and a ligand that stimulates a co-stimulatorymolecule on the surface of the T cells. In particular, T cellpopulations may be stimulated as described herein, such as by contactwith an anti-CD3 antibody, or antigen-binding fragment thereof, or ananti-CD2 antibody immobilized on a surface, or by contact with a proteinkinase C activator (e.g., bryostatin) in conjunction with a calciumionophore. For co-stimulation of an accessory molecule on the surface ofthe T cells, a ligand that binds the accessory molecule is used. Forexample, a population of T cells can be contacted with an anti-CD3antibody and an anti-CD28 antibody, under conditions appropriate forstimulating proliferation of the T cells. To stimulate proliferation ofeither CD4⁺ T cells or CD8⁺ T cells, an anti-CD3 antibody and ananti-CD28 antibody. Examples of an anti-CD28 antibody include 9.3, B-T3,XR-CD28 (Diaclone, Besancon, France) can be used as can other methodscommonly known in the art (Berg et al., Transplant Proc.30(8):3975-3977, 1998; Haanen et al., J. Exp. Med. 190(9):13191328,1999; Garland et al., J. Immunol Meth. 227(1-2):53-63, 1999).

In certain embodiments, the primary stimulatory signal and theco-stimulatory signal for the T cell may be provided by differentprotocols. For example, the agents providing each signal may be insolution or coupled to a surface. When coupled to a surface, the agentsmay be coupled to the same surface (i.e., in “cis” formation) or toseparate surfaces (i.e., in “trans” formation). Alternatively, one agentmay be coupled to a surface and the other agent in solution. In oneembodiment, the agent providing the co-stimulatory signal is bound to acell surface and the agent providing the primary activation signal is insolution or coupled to a surface. In certain embodiments, both agentscan be in solution. In another embodiment, the agents may be in solubleform, and then cross-linked to a surface, such as a cell expressing Fcreceptors or an antibody or other binding agent which will bind to theagents. In this regard, see for example, U.S. Patent ApplicationPublication Nos. 20040101519 and 20060034810 for artificial antigenpresenting cells (aAPCs) that are contemplated for use in activating andexpanding T cells in the present invention.

In one embodiment, the two agents are immobilized on beads, either onthe same bead, i.e., “cis,” or to separate beads, i.e., “trans.” By wayof example, the agent providing the primary activation signal is ananti-CD3 antibody or an antigen-binding fragment thereof and the agentproviding the co-stimulatory signal is an anti-CD28 antibody orantigen-binding fragment thereof; and both agents are co-immobilized tothe same bead in equivalent molecular amounts. In one embodiment, a 1:1ratio of each antibody bound to the beads for CD4⁺ T cell expansion andT cell growth is used. In certain aspects of the present invention, aratio of anti CD3:CD28 antibodies bound to the beads is used such thatan increase in T cell expansion is observed as compared to the expansionobserved using a ratio of 1:1. In one particular embodiment an increaseof from about 1 to about 3 fold is observed as compared to the expansionobserved using a ratio of 1:1. In one embodiment, the ratio of CD3:CD28antibody bound to the beads ranges from 100:1 to 1:100 and all integervalues there between. In one aspect of the present invention, moreanti-CD28 antibody is bound to the particles than anti-CD3 antibody,i.e., the ratio of CD3:CD28 is less than one. In certain embodiments ofthe invention, the ratio of anti CD28 antibody to anti CD3 antibodybound to the beads is greater than 2:1. In one particular embodiment, a1:100 CD3:CD28 ratio of antibody bound to beads is used. In anotherembodiment, a 1:75 CD3:CD28 ratio of antibody bound to beads is used. Ina further embodiment, a 1:50 CD3:CD28 ratio of antibody bound to beadsis used. In another embodiment, a 1:30 CD3:CD28 ratio of antibody boundto beads is used. In one preferred embodiment, a 1:10 CD3:CD28 ratio ofantibody bound to beads is used. In another embodiment, a 1:3 CD3:CD28ratio of antibody bound to the beads is used. In yet another embodiment,a 3:1 CD3:CD28 ratio of antibody bound to the beads is used.

Ratios of particles to cells from 1:500 to 500:1 and any integer valuesin between may be used to stimulate T cells or other target cells. Asthose of ordinary skill in the art can readily appreciate, the ratio ofparticles to cells may depend on particle size relative to the targetcell. For example, small sized beads could only bind a few cells, whilelarger beads could bind many. In certain embodiments the ratio of cellsto particles ranges from 1:100 to 100:1 and any integer valuesin-between and in further embodiments the ratio comprises 1:9 to 9:1 andany integer values in between, can also be used to stimulate T cells.The ratio of anti-CD3- and anti-CD28-coupled particles to T cells thatresult in T cell stimulation can vary as noted above, however certainpreferred values include 1:100, 1:50, 1:40, 1:30, 1:20, 1:10, 1:9, 1:8,1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1,9:1, 10:1, and 15:1 with one preferred ratio being at least 1:1particles per T cell. In one embodiment, a ratio of particles to cellsof 1:1 or less is used. In one particular embodiment, a preferredparticle: cell ratio is 1:5. In further embodiments, the ratio ofparticles to cells can be varied depending on the day of stimulation.For example, in one embodiment, the ratio of particles to cells is from1:1 to 10:1 on the first day and additional particles are added to thecells every day or every other day thereafter for up to 10 days, atfinal ratios of from 1:1 to 1:10 (based on cell counts on the day ofaddition). In one particular embodiment, the ratio of particles to cellsis 1:1 on the first day of stimulation and adjusted to 1:5 on the thirdand fifth days of stimulation. In another embodiment, particles areadded on a daily or every other day basis to a final ratio of 1:1 on thefirst day, and 1:5 on the third and fifth days of stimulation. Inanother embodiment, the ratio of particles to cells is 2:1 on the firstday of stimulation and adjusted to 1:10 on the third and fifth days ofstimulation. In another embodiment, particles are added on a daily orevery other day basis to a final ratio of 1:1 on the first day, and 1:10on the third and fifth days of stimulation. One of skill in the art willappreciate that a variety of other ratios may be suitable for use in thepresent invention. In particular, ratios will vary depending on particlesize and on cell size and type.

In further embodiments of the present invention, the cells, such as Tcells, are combined with agent-coated beads, the beads and the cells aresubsequently separated, and then the cells are cultured. In analternative embodiment, prior to culture, the agent-coated beads andcells are not separated but are cultured together. In a furtherembodiment, the beads and cells are first concentrated by application ofa force, such as a magnetic force, resulting in increased ligation ofcell surface markers, thereby inducing cell stimulation.

By way of example, cell surface proteins may be ligated by allowingparamagnetic beads to which anti-CD3 and anti-CD28 are attached (3×28beads) to contact the T cells. In one embodiment the cells (for example,10⁴ to 10⁹ T cells) and beads (for example, DYNABEADS® M-450 CD3/CD28 Tparamagnetic beads at a ratio of 1:1) are combined in a buffer,preferably PBS (without divalent cations such as, calcium andmagnesium). Again, those of ordinary skill in the art can readilyappreciate any cell concentration may be used. For example, the targetcell may be very rare in the sample and comprise only 0.01% of thesample or the entire sample (i.e., 100%) may comprise the target cell ofinterest. Accordingly, any cell number is within the context of thepresent invention. In certain embodiments, it may be desirable tosignificantly decrease the volume in which particles and cells are mixedtogether (i.e., increase the concentration of cells), to ensure maximumcontact of cells and particles. For example, in one embodiment, aconcentration of about 2 billion cells/ml is used. In anotherembodiment, greater than 100 million cells/ml is used. In a furtherembodiment, a concentration of cells of 10, 15, 20, 25, 30, 35, 40, 45,or 50 million cells/ml is used. In yet another embodiment, aconcentration of cells from 75, 80, 85, 90, 95, or 100 million cells/mlis used. In further embodiments, concentrations of 125 or 150 millioncells/ml can be used. Using high concentrations can result in increasedcell yield, cell activation, and cell expansion. Further, use of highcell concentrations allows more efficient capture of cells that mayweakly express target antigens of interest, such as CD28-negative Tcells. Such populations of cells may have therapeutic value and would bedesirable to obtain in certain embodiments. For example, using highconcentration of cells allows more efficient selection of CD8+ T cellsthat normally have weaker CD28 expression.

In one embodiment of the present invention, the mixture may be culturedfor several hours (about 3 hours) to about 14 days or any hourly integervalue in between. In another embodiment, the mixture may be cultured for21 days. In one embodiment of the invention the beads and the T cellsare cultured together for about eight days. In another embodiment, thebeads and T cells are cultured together for 2-3 days. Several cycles ofstimulation may also be desired such that culture time of T cells can be60 days or more. Conditions appropriate for T cell culture include anappropriate media (e.g., Minimal Essential Media or RPMI Media 1640 or,X-vivo 15, (Lonza)) that may contain factors necessary for proliferationand viability, including serum (e.g., fetal bovine or human serum),interleukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, GM-CSF, IL-10, IL-12,IL-15, TGFβ, and TNF-α or any other additives for the growth of cellsknown to the skilled artisan. Other additives for the growth of cellsinclude, but are not limited to, surfactant, plasmanate, and reducingagents such as N-acetyl-cysteine and 2-mercaptoethanol. Media caninclude RPMI 1640, AIM-V, DMEM, MEM, α-MEM, F-12, X-Vivo 15, and X-Vivo20, Optimizer, with added amino acids, sodium pyruvate, and vitamins,either serum-free or supplemented with an appropriate amount of serum(or plasma) or a defined set of hormones, and/or an amount ofcytokine(s) sufficient for the growth and expansion of T cells.Antibiotics, e.g., penicillin and streptomycin, are included only inexperimental cultures, not in cultures of cells that are to be infusedinto a subject. The target cells are maintained under conditionsnecessary to support growth, for example, an appropriate temperature(e.g., 37° C.) and atmosphere (e.g., air plus 5% CO₂).

T cells that have been exposed to varied stimulation times may exhibitdifferent characteristics. For example, typical blood or apheresedperipheral blood mononuclear cell products have a helper T cellpopulation (T_(H), CD4⁺) that is greater than the cytotoxic orsuppressor T cell population (T_(C), CD8⁺). Ex vivo expansion of T cellsby stimulating CD3 and CD28 receptors produces a population of T cellsthat prior to about days 8-9 consists predominately of T_(H) cells,while after about days 8-9, the population of T cells comprises anincreasingly greater population of T_(C) cells. Accordingly, dependingon the purpose of treatment, infusing a subject with a T cell populationcomprising predominately of T_(H) cells may be advantageous. Similarly,if an antigen-specific subset of T_(C) cells has been isolated it may bebeneficial to expand this subset to a greater degree.

Further, in addition to CD4 and CD8 markers, other phenotypic markersvary significantly, but in large part, reproducibly during the course ofthe cell expansion process. Thus, such reproducibility enables theability to tailor an activated T cell product for specific purposes.

Therapeutic Application

The present invention encompasses a cell (e.g., T cell) modified toexpress a plurality of types of CARs, wherein each CAR combines anantigen recognition domain of a specific antibody with an intracellulardomain. In one embodiment, at least one of the plurality of types ofCARs comprises a primary signaling domain, such as CD3z or other primarysignaling domains detailed elsewhere herein. In one embodiment, at leastone of the plurality of types of CARs comprises a costimulatory domain.In one embodiment, the modified T cell expresses a plurality of types ofCARs that function as trans-signaling CARs, where T cell activation isdependent upon binding of more than one type of CAR to its targetedantigen. Therefore, in some instances, the modified T cell can elicit aCAR-mediated T-cell response. In one embodiment, the dependence of thebinding to more than one type of antigen allows the modified T cell toexhibit a heightened specificity to elicit a response upon binding of atumor cell rather than a normal bystander cell.

The invention encompasses a cell (e.g., T cell) modified to expresscostimulatory-only CARs, wherein each CAR combines an antigenrecognition domain of a specific antibody with an intracellular domainof for example, CD28, 4-1BB (CD137), or any combination thereof.Therefore, in some instances, the modified T cell can enhance endogenousT cell activation.

The invention provides the use of a plurality of types oftrans-signaling CARs to redirect the specificity of a primary T cell toa tumor antigen. Thus, the present invention also provides a method forstimulating a T cell-mediated immune response to a target cellpopulation or tissue in a mammal comprising the step of administering tothe mammal a T cell that expresses a plurality of types oftrans-signaling CARs, wherein each type of CAR comprises a bindingmoiety that specifically interacts with a predetermined target. In oneembodiment, the cell comprises a first CAR comprising a zeta chainportion comprising for example the intracellular domain of human CD3z,and a second CAR comprising a costimulatory signaling region.

In one embodiment, the trans-signaling CAR T cells of the invention canundergo robust in vivo T cell expansion and can persist for an extendedamount of time. In another embodiment, the CAR T cells of the inventionevolve into specific memory T cells that can be reactivated to inhibitany additional tumor formation or growth. For example, CAR T cells ofthe invention can undergo robust in vivo T cell expansion and persist athigh levels for an extended amount of time in blood and bone marrow andform specific memory T cells. Without wishing to be bound by anyparticular theory, CAR T cells may differentiate in vivo into a centralmemory-like state upon encounter and subsequent elimination of targetcells expressing the surrogate antigen.

Without wishing to be bound by any particular theory, the anti-tumorimmunity response elicited by the trans-signaling CAR-modified T cellsmay be an active or a passive immune response. In addition, thetrans-signaling CAR mediated immune response may be part of an adoptiveimmunotherapy approach in which CAR-modified T cells induce an immuneresponse specific to the antigen binding domain in the CAR.

In one embodiment, the present invention includes a type of cellulartherapy where T cells are genetically modified to express acostimulatory-only CAR and the T cell is infused to a recipient in needthereof. The infused cell is able to kill tumor cells in the recipient.Unlike antibody therapies, modified T cells are able to replicate invivo resulting in long-term persistence that can lead to sustained tumorcontrol. In one embodiment, the costimulatory-only CAR functions with anindependent receptor to elicit a T cell response. For example, theindependent receptor can include a CD3z-containing first generation CAR,an endogenous T cell receptor, or exogenous T cell receptor.

While the data disclosed herein specifically disclose trans-signalingCAR T cells expressing a first CAR comprising an anti-mesothelin scFvand CD3z and second CAR comprising an anti-FRa scFv and CD28, theinvention should be construed to include any number of variations foreach of the components of the construct as described elsewhere herein.That is, the invention includes the use of any antigen binding domainsin the CARs to generate a CAR-mediated T-cell response specific to theantigen binding domain. For example, the antigen binding domain in theCAR of the invention can target a tumor antigen for the purposes oftreat cancer.

Cancers that may be treated include tumors that are not vascularized, ornot yet substantially vascularized, as well as vascularized tumors. Thecancers may comprise non-solid tumors (such as hematological tumors, forexample, leukemias and lymphomas) or may comprise solid tumors. Types ofcancers to be treated with the CARs of the invention include, but arenot limited to, carcinoma, blastoma, and sarcoma, and certain leukemiaor lymphoid malignancies, benign and malignant tumors, and malignanciese.g., sarcomas, carcinomas, and melanomas. Adult tumors/cancers andpediatric tumors/cancers are also included.

Hematologic cancers are cancers of the blood or bone marrow. Examples ofhematological (or hematogenous) cancers include leukemias, includingacute leukemias (such as acute lymphocytic leukemia, acute myelocyticleukemia, acute myelogenous leukemia and myeloblastic, promyelocytic,myelomonocytic, monocytic and erythroleukemia), chronic leukemias (suchas chronic myelocytic (granulocytic) leukemia, chronic myelogenousleukemia, and chronic lymphocytic leukemia), polycythemia vera,lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and highgrade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavychain disease, myelodysplastic syndrome, hairy cell leukemia andmyelodysplasia.

Solid tumors are abnormal masses of tissue that usually do not containcysts or liquid areas. Solid tumors can be benign or malignant.Different types of solid tumors are named for the type of cells thatform them (such as sarcomas, carcinomas, and lymphomas). Examples ofsolid tumors, such as sarcomas and carcinomas, include fibrosarcoma,myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, and othersarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,rhabdomyosarcoma, colon carcinoma, lymphoid malignancy, pancreaticcancer, breast cancer, lung cancers, ovarian cancer, prostate cancer,hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma,adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma,papillary thyroid carcinoma, pheochromocytomas sebaceous glandcarcinoma, papillary carcinoma, papillary adenocarcinomas, medullarycarcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bileduct carcinoma, choriocarcinoma, Wilms' tumor, cervical cancer,testicular tumor, seminoma, bladder carcinoma, melanoma, and CNS tumors(such as a glioma (such as brainstem glioma and mixed gliomas),glioblastoma (also known as glioblastoma multiforme) astrocytoma, CNSlymphoma, germinoma, medulloblastoma, Schwannoma craniopharyogioma,ependymoma, pinealoma, hemangioblastoma, acoustic neuroma,oligodendroglioma, menangioma, neuroblastoma, retinoblastoma and brainmetastases).

In one embodiment, the antigen bind moiety portion of thetrans-signaling CAR T cells of the invention is designed to treat aparticular cancer. In one embodiment, the trans-signaling CAR T cells ofthe invention are modified to express a first CAR targeting a firstantigen and a second CAR targeting a second antigen, where the first andsecond antigen are expressed on a particular tumor or cancer. Forexample, the trans-signaling CAR T cells modified to target mesothelinand folate can be used to treat cancers and disorders including ovariancancer. In another embodiment, the trans-signaling CAR T cells aremodified to target EGFRvIII and IL-13Ra to treat glioma. In anotherembodiment, the trans-signaling CAR T cells are modified to target EGFRand CA-IX to treat renal cell carcinoma (RCC). In another embodiment,the trans-signaling CAR T cells are modified to target MUC1 and EGFR totreat pancreatic cancer. In another embodiment, the trans-signaling CART cells are modified to target mesothelin and HER2 to treat breastcancer.

In another embodiment, a CAR of the invention can be designed to targetCD22 to treat diffuse large B-cell lymphoma.

In one embodiment, cancers and disorders include but are not limited topre-B ALL (pediatric indication), adult ALL, mantle cell lymphoma,diffuse large B-cell lymphoma, salvage post allogenic bone marrowtransplantation, and the like can be treated using a combination of CARsthat target CD19, CD20, CD22, and ROR1.

In one embodiment, a CAR of the invention can be designed to targetmesothelin to treat mesothelioma, pancreatic cancer, ovarian cancer, andthe like. In another embodiment, a CAR of the invention can be designedto target CD33/IL3Ra to treat acute myelogenous leukemia and the like.In a further embodiment, a CAR of the invention can be designed totarget c-Met to treat triple negative breast cancer, non-small cell lungcancer, and the like.

In one embodiment, a CAR of the invention can be designed to target PSMAto treat prostate cancer and the like. In another embodiment, a CAR ofthe invention can be designed to target Glycolipid F77 to treat prostatecancer and the like. In a further embodiment, a CAR of the invention canbe designed to target EGFRvIII to treat glioblastoma and the like.

In one embodiment, a CAR of the invention can be designed to target GD-2to treat neuroblastoma, melanoma, and the like. In another embodiment, aCAR of the invention can be designed to target NY-ESO-1 TCR to treatmyeloma, sarcoma, melanoma, and the like. In a further embodiment, a CARof the invention can be designed to target MAGE A3 TCR to treat myeloma,sarcoma, melanoma, and the like.

However, the invention should not be construed to be limited to solelyto the antigen targets and diseases disclosed herein. Rather, theinvention should be construed to include any antigenic target that isassociated with a disease where a CAR can be used to treat the disease.

The CAR-modified T cells of the invention may also serve as a type ofvaccine for ex vivo immunization and/or in vivo therapy in a mammal.Preferably, the mammal is a human.

With respect to ex vivo immunization, at least one of the followingoccurs in vitro prior to administering the cell into a mammal: i)expansion of the cells, ii) introducing a nucleic acid encoding a CAR tothe cells, and/or iii) cryopreservation of the cells.

Ex vivo procedures are well known in the art and are discussed morefully below. Briefly, cells are isolated from a mammal (preferably ahuman) and genetically modified (i.e., transduced or transfected invitro) with a vector expressing a CAR disclosed herein. The CAR-modifiedcell can be administered to a mammalian recipient to provide atherapeutic benefit. The mammalian recipient may be a human and theCAR-modified cell can be autologous with respect to the recipient.Alternatively, the cells can be allogeneic, syngeneic or xenogeneic withrespect to the recipient.

The procedure for ex vivo expansion of hematopoietic stem and progenitorcells is described in U.S. Pat. No. 5,199,942, incorporated herein byreference, can be applied to the cells of the present invention. Othersuitable methods are known in the art, therefore the present inventionis not limited to any particular method of ex vivo expansion of thecells. Briefly, ex vivo culture and expansion of T cells comprises: (1)collecting CD34+ hematopoietic stem and progenitor cells from a mammalfrom peripheral blood harvest or bone marrow explants; and (2) expandingsuch cells ex vivo. In addition to the cellular growth factors describedin U.S. Pat. No. 5,199,942, other factors such as flt3-L, IL-1, IL-3 andc-kit ligand, can be used for culturing and expansion of the cells.

In addition to using a cell-based vaccine in terms of ex vivoimmunization, the present invention also provides compositions andmethods for in vivo immunization to elicit an immune response directedagainst an antigen in a patient.

Generally, the cells activated and expanded as described herein may beutilized in the treatment and prevention of diseases that arise inindividuals who are immunocompromised. In particular, the CAR-modified Tcells of the invention are used in the treatment of cancer. In certainembodiments, the cells of the invention are used in the treatment ofpatients at risk for developing cancer. Thus, the present inventionprovides methods for the treatment or prevention of cancer comprisingadministering to a subject in need thereof, a therapeutically effectiveamount of the CAR-modified T cells of the invention.

The CAR-modified T cells of the present invention may be administeredeither alone, or as a pharmaceutical composition in combination withdiluents and/or with other components such as IL-2 or other cytokines orcell populations. Briefly, pharmaceutical compositions of the presentinvention may comprise a target cell population as described herein, incombination with one or more pharmaceutically or physiologicallyacceptable carriers, diluents or excipients. Such compositions maycomprise buffers such as neutral buffered saline, phosphate bufferedsaline and the like; carbohydrates such as glucose, mannose, sucrose ordextrans, mannitol; proteins; polypeptides or amino acids such asglycine; antioxidants; chelating agents such as EDTA or glutathione;adjuvants (e.g., aluminum hydroxide); and preservatives. Compositions ofthe present invention are preferably formulated for intravenousadministration.

Pharmaceutical compositions of the present invention may be administeredin a manner appropriate to the disease to be treated (or prevented). Thequantity and frequency of administration will be determined by suchfactors as the condition of the patient, and the type and severity ofthe patient's disease, although appropriate dosages may be determined byclinical trials.

When “an immunologically effective amount”, “an anti-tumor effectiveamount”, “an tumor-inhibiting effective amount”, or “therapeutic amount”is indicated, the precise amount of the compositions of the presentinvention to be administered can be determined by a physician withconsideration of individual differences in age, weight, tumor size,extent of infection or metastasis, and condition of the patient(subject). It can generally be stated that a pharmaceutical compositioncomprising the T cells described herein may be administered at a dosageof 10⁴ to 10⁹ cells/kg body weight, preferably 10⁵ to 10⁶ cells/kg bodyweight, including all integer values within those ranges. T cellcompositions may also be administered multiple times at these dosages.The cells can be administered by using infusion techniques that arecommonly known in immunotherapy (see, e.g., Rosenberg et al., New Eng.J. of Med. 319:1676, 1988). The optimal dosage and treatment regime fora particular patient can readily be determined by one skilled in the artof medicine by monitoring the patient for signs of disease and adjustingthe treatment accordingly.

In certain embodiments, it may be desired to administer activated Tcells to a subject and then subsequently redraw blood (or have anapheresis performed), activate T cells therefrom according to thepresent invention, and reinfuse the patient with these activated andexpanded T cells. This process can be carried out multiple times everyfew weeks. In certain embodiments, T cells can be activated from blooddraws of from 10 cc to 400 cc. In certain embodiments, T cells areactivated from blood draws of 20 cc, 30 cc, 40 cc, 50 cc, 60 cc, 70 cc,80 cc, 90 cc, or 100 cc. Not to be bound by theory, using this multipleblood draw/multiple reinfusion protocol may serve to select out certainpopulations of T cells.

The administration of the subject compositions may be carried out in anyconvenient manner, including by aerosol inhalation, injection,ingestion, transfusion, implantation or transplantation. Thecompositions described herein may be administered to a patientsubcutaneously, intradermally, intratumorally, intranodally,intramedullary, intramuscularly, by intravenous (i.v.) injection, orintraperitoneally. In one embodiment, the T cell compositions of thepresent invention are administered to a patient by intradermal orsubcutaneous injection. In another embodiment, the T cell compositionsof the present invention are preferably administered by i. v. injection.The compositions of T cells may be injected directly into a tumor, lymphnode, or site of infection.

In certain embodiments of the present invention, cells activated andexpanded using the methods described herein, or other methods known inthe art where T cells are expanded to therapeutic levels, areadministered to a patient in conjunction with (e.g., before,simultaneously or following) any number of relevant treatmentmodalities, including but not limited to treatment with agents such asantiviral therapy, cidofovir and interleukin-2, Cytarabine (also knownas ARA-C) or natalizumab treatment for MS patients or efalizumabtreatment for psoriasis patients or other treatments for PML patients.In further embodiments, the T cells of the invention may be used incombination with chemotherapy, radiation, immunosuppressive agents, suchas cyclosporin, azathioprine, methotrexate, mycophenolate, and FK506,antibodies, or other immunoablative agents such as CAM PATH, anti-CD3antibodies or other antibody therapies, cytoxin, fludaribine,cyclosporin, FK506, rapamycin, mycophenolic acid, steroids, FR901228,cytokines, and irradiation. These drugs inhibit either the calciumdependent phosphatase calcineurin (cyclosporine and FK506) or inhibitthe p70S6 kinase that is important for growth factor induced signaling(rapamycin) (Liu et al., Cell 66:807-815, 1991; Henderson et al., Immun.73:316-321, 1991; Bierer et al., Curr. Opin. Immun. 5:763-773, 1993). Ina further embodiment, the cell compositions of the present invention areadministered to a patient in conjunction with (e.g., before,simultaneously or following) bone marrow transplantation, T cellablative therapy using either chemotherapy agents such as, fludarabine,external-beam radiation therapy (XRT), cyclophosphamide, or antibodiessuch as OKT3 or CAMPATH. In another embodiment, the cell compositions ofthe present invention are administered following B-cell ablative therapysuch as agents that react with CD20, e.g., Rituxan. For example, in oneembodiment, subjects may undergo standard treatment with high dosechemotherapy followed by peripheral blood stem cell transplantation. Incertain embodiments, following the transplant, subjects receive aninfusion of the expanded immune cells of the present invention. In anadditional embodiment, expanded cells are administered before orfollowing surgery.

The dosage of the above treatments to be administered to a patient willvary with the precise nature of the condition being treated and therecipient of the treatment. The scaling of dosages for humanadministration can be performed according to art-accepted practices.Strategies for CAR T cell dosing and scheduling have been discussed(Ertl et al, 2011, Cancer Res, 71:3175-81; Junghans, 2010, Journal ofTranslational Medicine, 8:55).

EXPERIMENTAL EXAMPLES

The invention is further described in detail by reference to thefollowing experimental examples. These examples are provided forpurposes of illustration only, and are not intended to be limitingunless otherwise specified. Thus, the invention should in no way beconstrued as being limited to the following examples, but rather, shouldbe construed to encompass any and all variations which become evident asa result of the teaching provided herein.

Without further description, it is believed that one of ordinary skillin the art can, using the preceding description and the followingillustrative examples, make and utilize the compounds of the presentinvention and practice the claimed methods. The following workingexamples therefore, specifically point out the preferred embodiments ofthe present invention, and are not to be construed as limiting in anyway the remainder of the disclosure.

Example 1 Chimeric Antigen Receptor T Cells with Dissociated SignalingDomains Exhibit Focused Anti-Tumor Activity In Vivo

By way of example, a trans-signaling CAR strategy was utilized tospecify T cells for robust effector function that is selective for tumorbut not normal tissue, whereby the T cell activation signal 1 (e.g.,CD3z module) is physically dissociated from the costimulatory signal 2(e.g., CD28 module) in two CARs of differing antigen specificity; oneagainst mesothelin and one against □-folate receptor (aFR).

For example, human T cells were genetically modified to co-expresssignal 1 (Anti-Meso scFv-CD3z) and signal 2 (Anti-aFR scFv-CD28) CARs intrans. Trans-signaling CART cells showed weak cytokine secretion againsttumor targets expressing only one tumor associated antigen (TAA),similar to first generation CART cells bearing CD3ζ only, butdemonstrated enhanced cytokine secretion and resistance to antigeninduced cell death (AICD) upon encountering natural or engineered tumorcells co-expressing mesothelin and aFR antigens, equivalent to that ofsecond generation CART cells with dual signaling in cis. Importantly,CART cells with dual specificity showed potent anti-cancer activity,tumor localization and persistence in vivo which was superior to firstgeneration CART cells and equivalent to second generation CARs. However,second generation CART cells also exhibited potent activity againstengineered targets expressing mesothelin alone to recapitulate normaltissue, whereas trans-signaling CART cells did not. Thus, a dualspecificity, trans-signaling CAR approach may potentiate the therapeuticefficacy of CART cells while minimizing activity against normal tissues.

To diminish “on target” toxicity and improve tumor-focused targeting andattack, the concept of a trans-signaling CAR strategy where the T cellactivation signal 1 (CD3z module) is physically dissociated from thecostimulatory signal 2 (CD28 module) was developed and tested. Since aFRand mesothelin are TAAs co-expressed in the vast majority of epithelialovarian cancers but are expressed differentially and at low levels innormal tissues (Chang et al., 1992, Int. J. Cancer 50:373-381; Kalli etal., 2008, Gynecol. Oncol. 108:619-626; Toffoli et al., 1997, Int. J.Cancer 74:193-198; Parker et al., 2005, Anal. Biochem. 338:284-293;Mantovani et al., 1994, Eur. J. Cancer 30A (363-369)), two independentCARs of distinct specificity were utilized; a signal 1 CAR (Meso-CD3zonly), and a signal 2 CAR (aFR-CD28 only) using pre-validated scFvs(Lanitis et al., 2012, Mol. Ther. 20:633-643; Song et al., 2011, CancerRes. 71:4617-4627). In this fashion, T cells transduced to co-expressboth CARs can exhibit potent in vitro and in vivo effector functionsthat are driven by tumor encounter and coupled with diminished damage tonormal tissues.

The materials and methods employed in these experiments are nowdescribed.

CAR Constructs

First and second generation anti-mesothelin M CAR constructs designatedP4-z and M-28z have been previously described (Lanitis et al., 2012,Mol. Ther. 20:633-643). The anti-FRa CAR construct designated as F-28 isa derivative of a previously characterized CAR (Song et al., 2011,Cancer Res. 71:4617-4627) that encompasses the MOV-19 scFv driven by theEF1a promoter and fused only to the CD28 intracellular costimulatorysignaling region via CD8a hinge and CD28 transmembrane domain. Togenerate the F-28 CAR construct the M-28z CAR construct was used as atemplate for the PCR amplification of a 350-bp fragment containing CD8ahinge-CD28TM (transmembrane domain)-CD28ICD (intracellular domain) usingthe following primers: 5′- ACGC GCTAGCACCACGACGCCAGCGC-3′ (SEQ ID NO: 1;NheI is underlined) and 5′- ACGCGTCGACTTAGGAGCGATAGGCTGCGAAGTCGC -3′(SEQ ID NO: 2; SalI is underlined). The resulting PCR product containinga NheI site on the 5′- end and a SalI site on the 3′- end was digestedwith the relevant enzymes. Then the F-28z CAR construct (pELNSMOV19-28z) previously described was digested with the NheI and SalIrestriction enzymes to remove the CD8hinge-CD28TM-CD28ICD-CD3z andcreate compatible cohesive ends followed by gel purified. Next thedigested PCR product was ligated into the digested F-28z vector. Theresulting product containing the anti-FRa (F) MOV19 scFv followed byCD8ahinge-CD28TM-CD28ICD was designated M-z/F-28.

Recombinant Lentivirus Production

High-titer replication-defective lentiviral vectors were produced andconcentrated as previously described (Lanitis et al., 2012, Mol. Ther.20:633-643). 293T human embryonic kidney cells were seeded at 10×10⁶ perT-150 tissue culture flask 24 h before transfection. All plasmid DNAwere purified using the QIAGEN Endo-free Maxi prep kit. Cells weretransfected with 7 ug pVSV-G (VSV glycoprotein expression plasmid), 18μg pRSV.REV (Rev expression plasmid), 18 μg of pMDLg/p.RRE (Gag/Polexpression plasmid), and 15 μg of pELNS transfer plasmid using ExpressInn (Open Biosytems). The viral supernatant was harvested at 24 and 48 hpost-transfection. Viral particles were concentrated and resuspended in0.4 ml by ultracentrifugation for 3 h at 25,000 rpm with a Beckman SW28rotor (Beckman Coulter, Fullerton, Calif.).

Human T Cell Transduction

Primary human T cells were isolated from healthy volunteer donorsfollowing leukapheresis by negative selection. T cells were cultured incomplete media (RPMI 1640 supplemented with 10% heat inactivated fetalbovine serum (FBS), 100 U/ml penicillin, 100 μg/ml streptomycin sulfate,10-mM HEPES), and stimulated with anti-CD3 and anti-CD28 mAbs coatedbeads (Invitrogen, Grand Island, N.Y.) as described (Levine et al.,1997, J. Immunol. 159:5921-5930). 12-24 hr after activation, T cellswere transduced with lentiviral vectors at MOI of ˜5-10. CD4⁺ and CD8⁺ Tcells used for in vivo experiments were mixed at 1:1 ratio, activated,and transduced. Human recombinant interleukin-2 (Novartis) was addedevery other day to a 50 IU/ml final concentration. Cell density of0.5-1×10⁶ cells/ml was maintained. Rested engineered T cells wereadjusted for identical transgene expression prior to functional assays.

Cell Lines

Lentivirus packaging was performed in the immortalized normal fetalrenal 293T cell line purchased from ATCC. Human cell lines used inimmune based assays include the established human ovarian cancer celllines A1847 and C30. For bioluminescence assays, target cancer celllines were transfected to express firefly luciferase (fLuc), enriched byantibiotic selection positive expression by bioluminescence imaging. Thehuman tumor cell line, C30, was transduced with lentivirus to expresshuman mesothelin (C30-M) or human aFR (C30-F) or both (C30M/F). 293Tcells and tumor cell lines were maintained in RPMI-1640 (Invitrogen,Grand Island, N.Y.) supplemented with 10% (v/v) heat-inactivated FBS, 2mM L-glutamine, and 100 μg/mL penicillin and 100 U/mL streptomycin. Allcell lines were routinely tested for mycoplasma contamination.

Cytokine Release Assays

Cytokine release assays were performed by coculture of 1×10⁵T cells with1×10⁵target cells per well in triplicate in 96-well round bottom platesin a final volume of 200 ul of T cell media. After 20˜24 hr, co-culturesupernatants were assayed for presence of IFN-g using an ELISA Kit,according to manufacturer's instructions (Biolegend, San Diego, Calif.).Values represent the mean of triplicate wells. IL-2, IL-4, IL-10, TNF-aand MIP-1a cytokines were measured by flow cytometry using Cytokine BeadArray, according to manufacturer's instructions (BD Biosciences, SanJose, Calif.).

Cytotoxicity Assays

⁵¹Cr release assays were performed as described (Johnson et al., 2006,J. Immunol. 177:6548-6559). Target cells were labeled with 100 uCi 100uCi ⁵¹Cr at 37° C. for 1.5 hours. Target cells were washed three timesin PBS, resuspended in CM at 10⁵ viable cells/mL and 100 uL added perwell of a 96-well V-bottom plate. Effector cells were washed twice in CMand added to wells at the given ratios. Plates were quickly centrifugedto settle cells, and incubated at 37° C. in a 5% CO₂ incubator for 4 or18 hours after which time the supernatants were harvested, transferredto a lumar-plate (Packard, Pangbourne, UK) and counted using a 1450Microbeta Liquid Scintillation Counter (Perkin-Elmer). For the bystandercytotoxicity assays, ⁵¹Cr labeled mesothelin-negative target cells weremixed with unlabelled mesothelin-positive targets cells at a ratio 1:1for a final concentration of 10⁵ viable cells/ml before being incubatedwith the effector T cells at the given ratios. Spontaneous ⁵¹Cr releasewas evaluated in target cells incubated with medium alone. Maximal ⁵¹Crrelease was measured in target cells incubated with SDS at a finalconcentration of 2% (v/v). Percent specific lysis was calculated as(experimental−spontaneous lysis/maximal−spontaneous lysis) times 100.

Xenograft Model of Ovarian Cancer

Six to 12-week-old NOD/SCID/γ-chain−/− (NSG) mice were bred, treated andmaintained under pathogen-free conditions. For an established ovariancancer model, 6 to 12-week-old female NSG mice were inoculated s.c. with5×10⁶ A1847 fLuc+ cells on the flank on day 0. For the bilateral mousemodel 5×10⁶ of A1847M⁺/F⁺ and A1847M⁺/F⁻ cells were inoculated s.c.separately in the same NSG mice on opposite hind flanks. After tumorswere established at about 7 weeks, human primary T cells were activated,and transduced as described above. After 2 weeks T cell expansion, whenthe tumor burden was 250-350 mm³, mice were i.v injected with T cells.Tumor dimensions were measured with calipers, and tumor volumescalculated using the formula V=1/2(length×width²), where length isgreatest longitudinal diameter and width is greatest transversediameter. Animals were imaged prior to T cell transfer and about everyweek thereafter to evaluate tumor growth. Photon emission from fLuc+cells was quantified using the “Living Image” software (Xenogen,Alameda, Calif.) for all in vivo experiments.

Bioluminescence Imaging

Tumor growth was also monitored by Bioluminescent imaging (BLI). BLI wasperformed using Xenogen IVIS imaging system and the photons emitted fromfLuc-expressing cells within the animal body were quantified usingLiving Image software (Xenogen, Alameda, Calif.). Briefly, mice bearingA1847 fLuc⁺ tumor cells were injected intraperitoneally with D-luciferin(150 mg/kg stock, 100 μL of D-luciferin per 10 grams of mouse bodyweight) suspended in PBS and imaged under isoflurane anesthesia after5-10 minutes. A pseudocolor image representing light intensity (blue,least intense; red, most intense) was generated using Living Image. BLIfindings were confirmed at necropsy.

Flow Cytometric Analysis

The following MAbs were used for phenotypic analysis: PE mouseanti-Human CD3; FITC anti-human CD4; APC anti-human CD8; PE-anti-humanCD45, APC-Cy7 anti-human CD69, Alexa 647 anti-human CD107a and Alexa 647anti-human CD107b. 7-AAD was used for viability staining. All mAbs werepurchased from BD Biosciences, San Jose, Calif. In T cell transferexperiments, peripheral blood was obtained via retro-orbital bleedingand stained for the presence of human CD45, CD4, and CD8 T cells. Aftergating on the human CD45⁺ population, the CD4⁺ and CD8⁺ subsets werequantified using TruCount tubes (BD Biosciences, San Jose, Calif.) withknown numbers of fluorescent beads as described in the manufacturer'sinstructions. Tumor cell surface expression of mesothelin was performedusing soluble P4 anti-mesothelin scFv followed by PE-labeledstreptavidin. T cell surface expression of the M-CAR was evaluated usingV5-tagged recombinant mesothelin followed by Alexa-647 conjugatedanti-V5 tag or biotinylated streptavidin. F-CAR expression was evaluatedusing GFP as a reporter gene. Acquisition and analysis was performedusing a BD FACS CANTO II with DIVA software.

Immunohistochemistry

Mice were euthanized by CO2 inhalation and tumors were collected inTissue-Tek O.C.T. Compound, and frozen at −80 oC. A standardStrept-avidin horseradish immunoperoxidase method was used for human CD3staining. Primary and secondary antibodies were diluted in buffercontaining 10% normal goat serum. 7 μm cryosections were fixed in coldacetone for 5 min at 4° C. and blocked with Dako's (Carpentaria, Calif.)peroxidase blocking system for 10 minutes. Sequential incubationsincluded the following: 10% normal goat serum (30 min at RT); primaryrabbit anti-human CD3 monoclonal antibody (Thermo Scientific RM-9107) at1:100 dilution (45 min. at RT); secondary biotinylated goat anti-rabbitantibody at 1:200 dilution (30 min at RT); strept-avidin-biotinylatedhorseradish peroxidase complex reagent (Dako) (30 min at RT); and three5 minute washes in buffer after each incubations. Sections were thenexposed to the chromagen DAB plus from Dako for 5 min at RT andcounterstained with hematoxylin, dehydrated, cleared and mounted. Forthe quantification of CD3⁺ T cells within the tumors, T cells werecounted in 10 randomly selected intratumoral fields of each slide athigh magnification (20×).

Apoptosis Assay

CAR T cells (3×10⁵) were co-cultured with equal number of tumor cells(3×10⁵) for three days. After the co-culture period the cells werestained with CD3 Ab and recombinant mesothelin for CAR expression. Thenthe cells were washed with phosphate-buffered saline, and labeled withannexin V-FITC and 7AAD using an Apoptosis Detection Kit (BD Pharmingen)according to instructions from the manufacturer. Samples were run on aFACScan, and data were analyzed using Cellquest.

Degranulation Assay

The degranulation assay was performed as earlier described (Betts etal., 2003, J Immunol Methods, 281(1-2): 65-78) with minor modifications.Target cells (1×10⁵) were co-cultured with an equal number of effectorcells in 0.1 mL per well in a 96-well plate in triplicate. Control wellscontained either T cells alone. Anti-CD107a and Ab Anti-CD107b (10 ulper well) or IgG1 conjugated to FITC (BD Biosciences, San Jose, Calif.)were added in addition to lul/sample of monensin (BD Biosciences, SanJose, Calif.) and incubated for 4-5 h at 37° C. Cells were washed 2times with PBS, stained for expression of the P4 CAR, CD8 and CD69 andanalyzed on a FACS DIVA II.

Statistical Analysis

Statistical analysis was performed using two-way repeated measures ANOVAfor the tumor burden (tumor volume, photon counts). Student's t test wasused to evaluate differences in absolute numbers of transferred T cells,cytokine secretion and specific cytolysis. Kaplan-Meier survival curveswere compared using the log-rank test. GraphPad Prism 4.0 (GraphPadSoftware) was used for the statistical calculations. P<0.05 wasconsidered significant.

The results of the experiments are now described.

CAR Construction

Anti-mesothelin CAR constructs comprised of the P4 scFv linked to a CD8ahinge and transmembrane region, followed by a CD3-z signaling moietyalone (M-z) or in tandem with the CD28 intracellular signaling motifwere previously shown to confer specific mesothelin-redirected activityin vitro and in vivo (Lanitis et al., 2012, Mol. Ther. 20:633-643) (FIG.1A). The costimulatory only anti-aFR CAR (F-28) construct is comprisedof the MOv-19 scFv linked to a CD8a hinge and CD28 transmembrane region,followed by the CD28 intracellular signaling motif; the signalingdeficient F-Dz construct lacks functional signaling domains (FIG. 1A).Primary human T cells were efficiently transduced with the twoCAR-encoding lentiviral vectors with >40% dual transduced T cellsreproducibly expressing both CARs (FIG. 1B). CART cell populations wereadjusted to equivalent frequencies of anti-mesothelin CART cells(60-70%) by adding untransduced T cells for all functional assays.

Trans-Signaling CART Cells Exert Superior Antigen Specific CytokineSecretion In Vitro

To evaluate the in vitro effector functions of CART cells in response tocells that express mesothelin alone versus tumor cells that co-expressmesothelin and aFR, TAA-negative C30 cancer cell lines were engineeredto over-express both antigens alone or together (FIG. 2A). T cellsengineered to express the M-z CAR recognized and secreted similar levelsof IFN-γ when co-cultured with C30 cells expressing either mesothelin(C30M) or mesothelin and aFR (C30M/F). Second generation M-28z CAR Tcells exerted superior IFN-γ secretion against all C30 cell variants. Incontrast, trans-signaling M-z/F-28 CART cells produced low levels ofIFN-γ against C30M similar to M-z CART cells. However, when exposed toC30M/F tumor cells, M-z/F-28 CART cells exerted enhanced IFN-gproduction. M-z/F-28 CAR activity against C30M/F cells also surpassedthat of M-z CART cells demonstrating that expression of both antigenspromotes the costimulation of M-z/F-28 CART cells through F-28 CAR.Consistent with this notion, co-expression of the M-z and thesignaling-deficient F-Dz CAR in T cells did not enhance their responseagainst C30-M/F cells. T cells expressing F-28 CAR alone in did notproduce any IFN-γ consistence with the absence of CD3-z signaling.Moreover, control T cells transduced to express green fluorescentprotein (GFP) or an anti-CD19 CAR containing CD3t with CD28 signalingmotifs in tandem (CD19-28z) (Milone et al., 2009, 17:4617-4627) did notproduce cytokines after stimulation with CD19-negative C30 cells,illustrating the need for antigen-specificity.

To test trans-signaling in a more clinically meaningful model, anovarian cancer cell line that naturally expresses both mesothelin andaFR, A1847 was employed (FIG. 2C). Trans-signaling CART cells secretedsignificantly higher levels of IFN-γ in response to A1847 compared toM-z CART cells, and similar to cis-signaling M-28z CART cells showingthat the natural expression of both antigens on tumor cells is capableof inducing costimulatory effects to trans-signaling CART cells (FIG.2D). IL-2 secretion from the dual transduced M-z/F-28 CART cells wassimilar to second generation P4-28z CART indicating that integrateddelivery of signal 1 and 2 in trans or cis can synergistically enhanceproduction of the latter cytokine (FIG. 2E).

Trans-Signaling CAR T Cells Show In Vitro Cytolytic Potency

Degranulation is a quantitative indicator of lytic function by T cells(Song et al., 2011, Cancer Res. 71:4617-1627). M-z and M-z/F-28 CAR CD8+T cells degranulated with similar upregulation of surface co-expressionof mobilized CD107 (Lysosomal-associated membrane protein 1) and theactivation-associated marker CD69 in response to C30M, but not whenstimulated with C30 (FIG. 3A). Consistent with active costimulation,M-28z CART cells displayed a superior cytolytic phenotype against C30Mcompared with M-z and M-z/F-28 CART cells. In contrast, exposure toA1847 tumor cells led to equivalent and elevated degranulation by cisM-28z and trans M-z/F-28 CART cells which was higher than by M-z CARTcells (FIG. 3A). GFP-T cells and anti-CD19 CART cells did notdegranulate in response to C30 or A1847. In chromium release assays, thecytolytic function by the various CART cell populations was similaragainst A1847 target cells although slightly higher release of ⁵¹Cr wasobserved in the M-z and M-28z co-cultures (FIG. 3B). In contrast, M-28zCART cells specifically lysed C30M cells at a higher level compared withM-z and M-z/F-28.

Trans-Signaling CAR T Cells are Resistant to AICD

Incorporation of costimulatory signaling regions into CARs has beenshown to increase resistance of CART cells to apoptosis upon activationwith tumors (Hombach et al. 2007, 56:731-737). To investigate ifprovision of CD28 costimulation in trans protects T cells fromantigen-induced cell death (AICD), single or dual CAR bearing T cellswere co-cultured with A1847 tumor cells (M⁺/F⁺) for three days and thenmeasured for their rate of apoptosis by staining the T cells with 7-AADand Annexin V. Apoptosis was elevated in M-z T cells exposed to A1847(34%) but reduced in trans-signalling M-z/F-28 CART cells (0.5-1%; FIG.4A). F-28 or control CD19-28z CART cells displayed no AICD, consistentwith the absence of antigenic stimulation. Consistent with past studies(Zhong et al., Mol. Ther. 2010, 18:413-420), cis-signaling M-28z CARTcells were also resistant to AICD (FIG. 4B).

Dual-Specific CAR T Cells Possess Enhanced In Vivo Anti-Tumor Potencyand Persistence

The capability of trans-signaling CART cells to inhibit human tumoroutgrowth was evaluated in vivo in immunodeficientNOD/SCID/IL-2R-γc^(hull) (NSG) mice inoculated s.c. with 1×10⁶ fireflyluciferase-expressing A1847 cells. Mice with established A1847 tumors(150-200 mm³) received intravenous injections of CART cells on days 55and 59 post-tumor inoculation. Tumor growth was modestly butsignificantly inhibited in mice receiving M-z CART cells (p=0.043),compared to saline, CD19-28z CART cells, F-28 CART cells or GFP-T cellcontrol groups 4 weeks after first T cell dose (FIG. 5A). Transfer ofcis M-28z or trans M-z/F-28 CART cells mediated similar andsignificantly better inhibition of tumor outgrowth (p=0.028) compared toM-z T cells indicating that incorporation of CD28 signaling domain incis or trans enhances the anti-tumor activity in vivo against tumorsco-expressing mesothelin and FRa TAAs (FIG. 5A). Measurement of thetumor-derived luciferase signaling from the treated mice confirmed thelower tumor burden in the mice treated with the trans-signaling CARTcells compared with first generation CART cells. Bioluminescence signalsfrom tumors of mice treated with trans- or cis-signaling CART cells wassimilar (FIG. 5B). Two weeks after first T cell dose, peripheral bloodCD8⁺ T and CD4⁺ T cell counts from mice injected with cis M-28z or transM-z/F-28 CART cells were similar and significantly higher than in theM-z group (p<0.05; FIG. 5C). No substantial human T cell persistence wasobserved in mice treated with CD19-28z CART, F-28 CART or GFP-T cells.

Trans-, But Not Cis-, Signaling CAR T Cells Exhibit More Limited In VivoActivity Against Cells Bearing Single Antigen

FRa-deficient A1847 cells were generated via transduction withlentiviral particles encoding for an shRNA specific for silencing aFRgene expression, as a surrogate for normal human mesothelial cellsexpressing only mesothelin. Fluorescence-activated cell sorting resultedin an enriched cancer cell population (˜98%) which lacked surface aFRexpression (A1847M⁺/F⁻) (FIG. 9). aFR expression was unaltered afterengineering cells with control shRNA (A1847M⁺/F⁺). No difference in thein vitro growth kinetic of A1847M⁺/F⁺ and A1847M⁺/F⁻ cells was observed.In co-culture assays, IFN-g secretion by trans M-z/F-28 CART cells wassignificantly reduced in response to A1847M⁺/F⁻ compared withA1847M⁺/F⁺, a confirmation of potent effector function only uponengagement of both antigens (FIG. 9B). Further, the level of reactivityby M-z/F-28 and M-z CART cells against A1847M⁺/F⁻ was not statisticallydifferent. Comparatively, cis M-28z CAR T cells secreted significantlyhigher amounts of IFN-g against A1847M⁺/F⁻, similar to that achievedwith A1847M⁺/F⁺.

To evaluate the in vivo potency of trans- or cis-signaling T cellsagainst A1847 cells expressing or lacking aFR, 5×10⁶ A1847M⁺/F⁺ andA1847M⁺/F⁻ cells were inoculated s.c. separately in the same NSG mice onopposite hind flanks. Mice with the two established A1847 (>330 mm³)tumors received tail vein injections of CART cells on days 45 and 49post-tumor inoculation and monitored for tumor outgrowth. Control ofA1847M⁺/F⁺ tumor outgrowth was identical between the trans M-z/F-28 CARTand cis-signaling M-28z CART cell groups (FIG. 6A). In contrast,inhibition of A1847M⁺/F⁻ outgrowth was partially but significantlyattenuated in the trans M-z/F-28 CART cell group compared with the cisM-28z mice group (p=0.0045; FIG. 6B). Further, trans-signaling CARTcells were statistically less effective in inhibiting the outgrowth ofA1847M⁺/F⁻, compared with their activity against the A1847M⁺/F⁺ tumor inthe same mice (p=0.0001; FIG. 6C). Bioluminescence imaging of the tumorsconfirmed these results (FIG. 6D).

Preferential Accumulation of Trans-Signaling CAR T Cells in Tumor InVivo

The accumulation of trans- and cis-signaling CART cells in regressingtumors from treated and euthanized mice with established masses in bothflanks was measured. Resected tumors from each group were harvested andsubjected to immunohistochemical analysis for the detection of humanCD3⁺ T cells (FIG. 7). The abundance of T cells in dual (A1847M⁺/F⁺) orsingle antigen expressing tumors (A1847M⁺/F⁻) from mice treated with cisM-28z CART cells was high and not statistically significantly different.In contrast, a significant increase in T cell accumulation was observedin tumors expressing both antigens compared to single antigen in micetreated with trans M-z/F-28 CART cells, illustrating selectivity. FewCD3⁺ T cells were detected in tumors resected at the same time from micethat received CD19-28z CART cells. Altogether these data show that thesurvival and accumulation of trans signaling CAR T cells into tumorsites is highly dependent upon engagement of FRa antigen to the FRaspecific-CAR for the optimal delivery of costimulation.

Trans-Signaling CAR Approach

Adoptive immunotherapy involving genetic modification of T cells withantigen-specific, chimeric, single-chain receptors is a promisingapproach for the treatment of cancer (Plaimauer et al., 2002, Blood,100:3626-3632). An important consideration for the therapeutic use ofadoptively transferred, gene-engineered T cells is whether they mayinduce extensive autoimmune damage to normal tissues expressing thetarget antigen (Ertl et al., 2011, Cancer Res. 71:3175-3181). The latterphenomenon has been observed in pre-clinical mouse models and clinicaltrials either using T cells genetically modified to express an exogenoustumor specific TCR or T cells redirected through the incorporation of achimeric antigen receptor (Heslop, 2010, Mol. Ther. 18:661-662). Mild tosevere autoimmune toxicity has been reported following transfer of tumorreactive T cells (Jena et al., 2010, Blood, 1035-1044; Zhong et al.,2010, Mol. Ther. 18:413-420) including liver toxicity in patients uponadoptive transfer of T cells gene-modified with an anti-CAIX scFvreceptor (Feugier et al., 2004, Blood 104:2675-2681) due to CAIXexpression on bile duct epithelium. Serious adverse events involvingdeath of a patient has been observed in to two distinct clinical trialsfollowing adoptive transfer of T cells gene-modified against CD19(Morgan et al., 2010, Cancer J. 16:336-341; Brentjens et al., 2010, Mol.Ther. 18:666-668) or T cell genetically modified to express anErbB2-specific CAR (Morgan et al., 2010, Cancer J. 16:336-341).Collectively, studies both in preclinical mouse models and in patientshave indicated that the number of T cells administered, the levels andlocation of antigen expressed on normal tissue, the type of signalingdomains incorporated into chimeric receptors, and the level of immunepreconditioning used must be carefully considered for use in engineeredT-cell therapy. (Ertl et al., 2011, Cancer Res. 71:3175-3181; Straathofet al., 2005, Blood 105:4247-4254; Cohen et al., 1999, Lymphoma34:473-480; Thomis et al., 2001, Blood, 97:1249-1257; Tey et al., 2007,Biol. Blood Marrow Transplant 13:913-924; Di Stasi et al., 2011, N.Engl. J. Med. 365:1673-1683; Kuo et al., 2010, Lab Chip 10:837-842).

Chimeric antigen receptors with one or more costimulatory signals confera new potential to respond to antigen with sustained proliferation andcytotoxicity, resistance to activation-induced cell death (AICD) andregulatory T-cell suppression (Song et al., 2011, Cancer Res.71:4617-4627; Emtage et al., 2008, Clin. Cancer Res. 8112-8122).However, the power of costimulation holds the potential for sustainedsurvival and activation against normal host tissues expressing lowlevels of the TAA. One approach to forestall this problem involves thephysical separation of signal 1 module (CD3ζ) from the signal 2 module(costimulation) through their incorporation into two distinct CARsspecific for two different antigens, to recapitulate natural T cellbiology and function. In this way, dual CART cells may selectivelytraffic, survive and exert sustained proliferation within the tumormicroenvironment since synergistic signals would be delivered to T-cellspreferentially at that location. Hence, the potential for “on-target”toxicity should be reduced commensurately. CARs can be engineered toprovide co-stimulation alone (Krause et al., 1998, J. Exp. Med.188:619-626). Furthermore, when jurkat cells are engineered toco-express hapten-specific CD3ζ- and CD28-based CARs, complementarysignaling can occur, leading to IL-2 production (Alvarez-Vallina et al.,Eur. J. Immunol. 26:2304-2309). Attempts to preferentially redirect CARTcell function against tumors expressing multiple antigens to limitpotential toxicity have been documented without much success orpotential for clinical application. One study attempted to enhance thespecificity of CART cells for tumor by endowing them with two firstgeneration CARs specific for ErbB2 and a-folate receptor.Dual-transduced T-cell populations expressed similar amounts of totalsurface CARs as mono-transduced T cells, but with lower expression ofeach individual CAR (Duong et al., 2011, Immunotherapy 3:33-48). Thisallowed for activity of dual transduced T cells against target cellsexpressing individual antigens being less than against tumor cellsexpressing both antigens. Since no costimulation is provided into theseCAR T cells due to the lack of costimulatory signaling regions in theindividual CARs themselves, they are unlikely to survive, persist andclear tumors in vivo. Another recent study proposed dual targeting ofErbB2 and MUC1 in breast cancer by generating dual CAR T cellstransduced with both a CD28 containing MUC1 CAR and a CD3t containingErbB2 CAR (Wilkie et al., 2010, Immunotherapy 4:365-367). In that study,dual CAR T cells were even less efficient than first generation CARTcells and failed to secrete IL-2 in response to trans-signaledcostimulation upon encounter with second antigen thus rendering thesystem not applicable for further pre-clinical investigation.

Here, two different CARs redirected against mesothelin and aFR wereutilized to test the concept of dissociated CAR signaling for selectiveanti-tumor activity (FIG. 10). The anti-mesothelin CAR contains the CD3signaling motif alone whereas the anti-aFR CAR includes only theintracellular domain of the CD28 costimulatory molecule. Propercostimulation of these engineered T cells relies upon the co-expressionof both aFR and mesothelin on the tumor cell surface. In comparisonsbetween trans-signaling CAR T cells with conventional first and secondgeneration anti-mesothelin CART cells, trans-signaling CART cells showedsimilar in vitro potency to cis-signaling CAR T cells and were capableof producing increased levels of Thl cytokines compared to firstgeneration CART cells. Notably, tumor-induced costimulation through theanti-aFR-28 CAR was capable of triggering enhanced secretion of IL-2, acytokine known to promote T cell proliferation and persistence in vivo(Kowolik et al., 2006, Cancer Res. 66:10995-11004; Sadelain et al.,2009, Curr. Opin. Immunol. 21:215-223). Furthermore, the costimulationdelivered in trans was sufficient to protect those CART cells from AICD,similar to the effects seen in cis-signaling second generation CARTcells (Song et al., 2012, Blood 119:696-706; Zhong et al., 2010, Mol.Ther. 18:413-420). The cytolytic potential of trans-signaling CART cellsin vitro was similar to first and second generation CART cells. This isconsistent with previous studies demonstrating no statisticallysignificant difference in specific tumor lysis by CAR T cells thatinclude or lack incorporated costimulatory signaling regions (Savoldo etal., 2011, J. Clin. Invest. 121:1822-1826; Song et al., 2011, CancerRes. 71:4617-4627; Hombach et al., 2001, Cancer Res. 61:1976-1982).

The significance of a trans-signaling CAR approach is best tested inusing preclinical models where the simultaneous targeting of human tumorcells and representative normal human tissue cells by CART cells can beevaluated. Ovarian cancers generally over express both aFR (90%) andmesothelin (70%), making this an attractive cancer type for study(Toffoli et al., 1997, Int. J. Cancer 74:193-198; Hassan et al., 2005,Appl. Immunohistochem. Mol. Mofphol. 13:243-247). Moreover, the patternof mesothelin and aFR expression on normal tissues is largelynon-overlapping. Mesothelin is expressed by normal mesothelial cellslining the pleura, peritoneum and peritoneum and at low levels byepithelial cells of the trachea, tonsils, fallopian tube and the testis(Chang et al., 1992, 50:373-381; Ordonez, 2003, Mod. Pathol.16:192-197). FRa expression is limited to the apical surface of theproximal tubules of the kidney, choroid plexus, lung epithelium, thyroidand intestinal brush border epithelial cells (Weitman et al., 1992,Cancer Res. 52:3396-3401; Holm et al., 1993, Adv. Exp. Med. Biol.338:757-760).

In this study, in vivo anti-tumor activity of trans-signaling CART cellswas initially tested by adoptively transferring the different CART cellpopulations into immunodeficient mice with established ovarian cancer,using the A1847 human ovarian cancer cell line expressing bothmesothelin and FRa. It was found that trans-signaling CART cells exhibitpotent antitumor activity against ovarian cancer in vivo, equivalent tothat achieved using conventional cis-signaling CAR T cells. Moreimportantly, the finding that trans-signaling CAR T cells are moreselective, potent and localized within cancers which bear both antigens,and comparatively spare normal cells bearing single antigen, may haveimportant translational ramifications. It has been recently shown thatmesothelin redirected T cells bearing an anti-mesothelin chimericantigen receptor and a chemokine receptor (CCR2) increased thelocalization of the genetically engineered T cells in malignant pleuralmesotheliomas secreting the chemokine CCL2 and thus improved thetherapeutic outcome in pre-clinical mouse models (Moon et al., 2011,Clin. Cancer Res. 17:4719-4730).

Upon trafficking to the normal tissues expressing mesothelin alone,trans-signaling CART cells receive only signal 1 upon antigen contact,provided through the grafted TCR CD3 domain. This can potentially renderthe designer T cells susceptible to AICD or drive them into anergy(Zhong et al., 2010, Mol. Ther. 18:413-420; Emtage et al., 2008, Clin.Cancer Res. 14:8112-8122; Berry et al., 2009, Tissue Antigens74:277-289) and restrict their potential for long term persistence andtumor elimination when applied to patients. Alternatively, recognitionof aFR alone and singular transmission of CD28 signal, in the absence ofmesothelin-directed CD3ζ-signals, does not activate trans-signaling CARTcells. Importantly, equal regression of tumors was observed in thepresent model using cis- or trans-signaling CAR T cells, illustratingthe idea that a dual CART cell approach can modestly improve safety forclinical application without significantly diminishing the antitumorpotential of the second generation CAR approach. Dual CART cells whichreceive combined CD3t and costimulatory signals upon antigen-specificinteractions with the tumor did not appear to be renderedcostimulation-independent and endowed with the capacity to reactstrongly against the tissue expressing single antigen. This was apparentin the present preclinical model where there was a significantdifference in the control of tumor bearing two antigens versus the morerapid growth of cells with only one antigen following treatment withtrans M-z/F-28 CART cells. This was further supported by thestatistically higher infiltration and accumulation of trans-signalingCART cells into the tumor sites where both antigens were expressed.

While trans-signaling CARs represent a novel approach to focus CARTcells to tumor cells and reduce their impact on singleantigen-expressing cells, this method is highly dependent uponidentification of two antigens which are nearly uniformly expressed in aparticular cancer type, with relatively low and non-overlappingexpression in normal tissues. Accordingly, the identification andapplication of alternative approaches to limit CART-mediated autoimmuneeffects remains warranted. One approach to limit such toxicity is thecareful design of a dose-escalation strategy to better define theoptimal T cell dose (Ertl et al., 2011, Cancer Res. 71:3175-3181). Someof the potential side effects of nontumor cell recognition by CART cellscan be overcome by the co-expression of conditional suicide genes suchas such as incorporation of HSV-TK or the cytoplasmic domain of Fas oran inducible caspase incorporated into genetically engineered T cells toabort any aberrant T-cell responses (Straathof et al., 2005, Blood105:4247-4254; Cohen et al., 1999, Leuk. Lymphoma 34:473-480; Thomis etal., 2001, Blood 97:1249-1257; Tey et al., 2007, Biol. Blood MarrowTransplant 13:913-924). Indeed the iCasp9 cell-suicide system has beenshown to increase the safety of cellular therapies in patients thatreceived T cells depleted of allo-reactive progenitor cells (Di Stasi etal., 2011, N. Engl. J. Med. 365:1673-1683). Furthermore electroporationof T cells with optimized RNAs encoding for CARs allows for transientCAR expression as a safety measure, has been proven effective inpreclinical mouse models and might bypass the associated safety concernsof integrating gene vectors (Zhao et al., 2010, Cancer Res.70:9053-9061). Notably, these approaches toward cell product safety arenot mutually exclusive and future application of dual CAR T cellsengineered with suicide switches as described elsewhere herein maybetter permit localized T cells accumulation within the tumormicroenvironment where they can preferentially exert enhanced anti-tumorpotency in a safe manner.

Example 2 CAR Constructs

P4-z (amino acid sequence) (SEQ ID NO: 3)MALPVTALLLPLALLLHAARPGSQVQLQQSGPGLVTPSQTLSLTCAISGDSVSSNSATWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRMSINPDTSKNQFSLQLNSVTPEDTAVYYCARGMMTYYYGMDVWGQGTTVTVSSGILGSGGGGSGGGGSGGGGSQPVLTQSSSLSASPGASASLTCTLRSGINVGPYRIYWYQQKPGSPPQYLLNYKSDSDKQQGSGVPSRFSGSKDASANAGVLLISGLRSEDEADYYCMIWHSSAAVFGGGTQLTVLSASTTTPAPRPPTPAPTIASRPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRP4-z (nucleotide sequence) (SEQ ID NO: 4)ATGGCCTTAC CAGTGACCGC CTTGCTCCTG CCGCTGGCCT TGCTGCTCCACGCCGCCAGG CCGGGATCTC AGGTACAGCT GCAGCAGTCA GGTCCAGGACTCGTGACGCC CTCGCAGACC CTCTCACTCA CCTGTGCCAT CTCCGGGGACAGTGTCTCTA GCAACAGTGC TACTTGGAAC TGGATCAGGC AGTCCCCATCGAGAGGCCTT GAGTGGCTGG GAAGGACATA CTACAGGTCC AAGTGGTATAACGACTATGC AGTATCTGTG AAAAGTCGAA TGAGCATCAA CCCAGACACATCCAAGAACC AGTTCTCCCT GCAGCTGAAC TCTGTGACTC CCGAGGACACGGCTGTGTAT TACTGTGCAA GAGGAATGAT GACTTACTAT TACGGTATGGACGTCTGGGG CCAAGGGACC ACGGTCACCG TCTCCTCAGG AATTCTAGGATCCGGTGGCG GTGGCAGCGG CGGTGGTGGT TCCGGAGGCG GCGGTTCTCAGCCTGTGCTG ACTCAGTCGT CTTCCCTCTC TGCATCTCCT GGAGCATCAGCCAGTCTCAC CTGCACCTTG CGCAGTGGCA TCAATGTTGG TCCCTACAGGATATACTGGT ACCAGCAGAA GCCAGGGAGT CCTCCCCAGT ATCTCCTGAACTACAAATCA GACTCAGATA AGCAGCAGGG CTCTGGAGTC CCCAGCCGCTTCTCTGGATC CAAAGATGCT TCGGCCAATG CAGGGGTTTT ACTCATCTCTGGGCTCCGGT CTGAGGATGA GGCTGACTAT TACTGTATGA TTTGGCACAGCAGCGCTGCT GTGTTCGGAG GAGGCACCCA ACTGACCGTC CTCTCCGCTAGCACCACGAC GCCAGCGCCG CGACCACCAA CACCGGCGCC CACCATCGCGTCGCGGCCCC TGTCCCTGCG CCCAGAGGCG TGCCGGCCAG CGGCGGGGGGCGCAGTGCAC ACGAGGGGGC TGGACTTCGC CTGTGATATC TACATCTGGGCGCCCTTGGC CGGGACTTGT GGGGTCCTTC TCCTGTCACT GGTTATCACCCTTTACTGCA GAGTGAAGTT CAGCAGGAGC GCAGACGCCC CCGCGTACCAGCAGGGCCAG AACCAGCTCT ATAACGAGCT CAATCTAGGA CGAAGAGAGGAGTACGATGT TTTGGACAAG AGACGTGGCC GGGACCCTGA GATGGGGGGAAAGCCGAGAA GGAAGAACCC TCAGGAAGGC CTGTACAATG AACTGCAGAAAGATAAGATG GCGGAGGCCT ACAGTGAGAT TGGGATGAAA GGCGAGCGCCGGAGGGGCAA GGGGCACGAT GGCCTTTACC AGGGTCTCAG TACAGCCACCAAGGACACCT ACGACGCCCT TCACATGCAG GCCCTGCCCC CTCGCTAAMOV19-28 (F-28) (amino acid sequence) (SEQ ID NO: 5)MALPVTALLLPLALLLHAARPGSSRAAQPAMAQVQLQQSGAELVKPGASVKISCKASGYSFTGYFMNWVKQSHGKSLEWIGRIHPYDGDTFYNQNFKDKATLTVDKSSNTAHMELLSLTSEDFAVYYCTRYDGSRAMDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIELTQSPASLAVSLGQRAIISCKASQSVSFAGTSLMHWYHQKPGQQPKLLIYRASNLEAGVPTRFSGSGSKTDFTLNIHPVEEEDAATYYCQQSREYPYTFGGGTKLEIKRAAASTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHY QPYAPPRDFAAYRSMOV19-28 (F-28) (nucleotide sequence) (SEQ ID NO: 6)ATGGCCTTAC CAGTGACCGC CTTGCTCCTG CCGCTGGCCT TGCTGCTCCACGCCGCCAGG CCGGGATCCT CTAGAGCGGC CCAGCCGGCC ATGGCCCAGGTGCAGCTGCA GCAGTCTGGA GCTGAGCTGG TGAAGCCTGG GGCTTCAGTGAAGATATCCT GCAAGGCTTC TGGTTACTCA TTTACTGGCT ACTTTATGAACTGGGTGAAG CAGAGCCATG GAAAGAGCCT TGAGTGGATT GGACGTATTCATCCTTACGA TGGTGATACT TTCTACAACC AGAACTTCAA GGACAAGGCCACATTGACTG TAGACAAATC CTCTAACACA GCCCACATGG AGCTCCTGAGCCTGACATCT GAGGACTTTG CAGTCTATTA TTGTACAAGA TACGACGGTAGTCGGGCTAT GGACTACTGG GGCCAAGGGA CCACGGTCAC CGTCTCCTCAGGTGGAGGCG GTTCAGGCGG AGGTGGCTCT GGCGGTGGCG GATCGGACATCGAGCTCACT CAGTCTCCAG CTTCTTTGGC TGTGTCTCTA GGGCAGAGGGCCATCATCTC CTGCAAGGCC AGCCAAAGTG TCAGTTTTGC TGGTACTAGTTTAATGCACT GGTACCACCA GAAACCAGGA CAGCAACCCA AACTCCTCATCTATCGTGCA TCCAACCTAG AAGCTGGGGT TCCTACCAGG TTTAGTGGCAGTGGGTCTAA GACAGACTTC ACCCTCAATA TCCATCCTGT GGAGGAGGAGGATGCTGCAA CCTATTACTG TCAGCAAAGT AGGGAATATC CGTACACGTTCGGAGGGGGG ACAAAGTTGG AAATAAAACG GGCGGCCGCTAGCACCACGACGCCAGCGCC GCGACCACCA ACACCGGCGC CCACCATCGC GTCGCAGCCCCTGTCCCTGC GCCCAGAGGC GTGCCGGCCA GCGGCGGGGG GCGCAGTGCACACGAGGGGG CTGGACTTCG CCTGTGATTT TTGGGTGCTG GTGGTGGTTGGTGGAGTCCT GGCTTGCTAT AGCTTGCTAG TAACAGTGGC CTTTATTATTTTCTGGGTGA GGAGTAAGAG GAGCAGGCTC CTGCACAGTG ACTACATGAACATGACTCCC CGCCGCCCCG GGCCCACCCG CAAGCATTAC CAGCCCTATGCCCCACCACG CGACTTCGCA GCCTATCGCT CCTA AMOV19-BB (F-BB) (amino acid sequence) (SEQ ID NO: 7)MALPVTALLLPLALLLHAARPGSSRAAQPAMAQVQLQQSGAELVKPGASVKISCKASGYSFTGYFMNWVKQSHGKSLEWIGRIHPYDGDTFYNQNFKDKATLTVDKSSNTAHMELLSLTSEDFAVYYCTRYDGSRAMDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIELTQSPASLAVSLGQRAIISCKASQSVSFAGTSLMHWYHQKPGQQPKLLIYRASNLEAGVPTRFSGSGSKTDFTLNIHPVEEEDAATYYCQQSREYPYTFGGGTKLEIKRAAASTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRF PEEEEGGCELMOV19-BB (F-BB) (nucleotide sequence) (SEQ ID NO: 8)ATGGCCTTAC CAGTGACCGC CTTGCTCCTG CCGCTGGCCT TGCTGCTCCACGCCGCCAGG CCGGGATCCT CTAGAGCGGC CCAGCCGGCC ATGGCCCAGGTGCAGCTGCA GCAGTCTGGA GCTGAGCTGG TGAAGCCTGG GGCTTCAGTGAAGATATCCT GCAAGGCTTC TGGTTACTCA TTTACTGGCT ACTTTATGAACTGGGTGAAG CAGAGCCATG GAAAGAGCCT TGAGTGGATT GGACGTATTCATCCTTACGA TGGTGATACT TTCTACAACC AGAACTTCAA GGACAAGGCCACATTGACTG TAGACAAATC CTCTAACACA GCCCACATGG AGCTCCTGAGCCTGACATCT GAGGACTTTG CAGTCTATTA TTGTACAAGA TACGACGGTAGTCGGGCTAT GGACTACTGG GGCCAAGGGA CCACGGTCAC CGTCTCCTCAGGTGGAGGCG GTTCAGGCGG AGGTGGCTCT GGCGGTGGCG GATCGGACATCGAGCTCACT CAGTCTCCAG CTTCTTTGGC TGTGTCTCTA GGGCAGAGGGCCATCATCTC CTGCAAGGCC AGCCAAAGTG TCAGTTTTGC TGGTACTAGTTTAATGCACT GGTACCACCA GAAACCAGGA CAGCAACCCA AACTCCTCATCTATCGTGCA TCCAACCTAG AAGCTGGGGT TCCTACCAGG TTTAGTGGCAGTGGGTCTAA GACAGACTTC ACCCTCAATA TCCATCCTGT GGAGGAGGAGGATGCTGCAA CCTATTACTG TCAGCAAAGT AGGGAATATC CGTACACGTTCGGAGGGGGG ACAAAGTTGG AAATAAAACG GGCGGCCGCTAGCACCACGACGCCAGCGCC GCGACCACCA ACACCGGCGC CCACCATCGC GTCGCAGCCCCTGTCCCTGC GCCCAGAGGC GTGCCGGCCA GCGGCGGGGG GCGCAGTGCACACGAGGGGG CTGGACTTCG CCTGTGATAT CTACATCTGG GCGCCCTTGGCCGGGACTTG TGGGGTCCTT CTCCTGTCAC TGGTTATCAC CCTTTACTGCAAACGGGGCA GAAAGAAACT CCTGTATATA TTCAAACAAC CATTTATGAGACCAGTACAA ACTACTCAAG AGGAAGATGG CTGTAGCTGC CGATTTCCAGAAGAAGAAGA AGGAGGATGT GAACTGTAAC6.5-BB (HER2-BB) (amino acid sequence) (SEQ ID NO: 9)MALPVTALLLPLALLLHAARPGSQVQLLQSGAELKKPGESLKISCKGSGYSFTSYWIAWVRQMPGKGLEYMGLIYPGDSDTKYSPSFQGQVTISVDKSVSTAYLQWSSLKPSDSAVYFCARHDVGYCSSSNCAKWPEYFQHWGQGTLVTVSSGGGGSGGGGSGGGGSQSVLTQPPSVSAAPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYGHTNRPAGVPDRFSGSKSGTSASLAISGFRSEDEADYYCAAWDDSLSGWVFGGGTKLTVLGASTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPE EEEGGCELC6.5-BB (HER2-BB) (nucleotide sequence) (SEQ ID NO: 10)ATGGCCTTAC CAGTGACCGC CTTGCTCCTG CCGCTGGCCT TGCTGCTCCACGCCGCCAGG CCGGGATCCC AGGTGCAGCT GTTGCAGTCT GGGGCAGAGTTGAAAAAACC CGGGGAGTCT CTGAAGATCT CCTGTAAGGG TTCTGGATACAGCTTTACCA GCTACTGGAT CGCCTGGGTG CGCCAGATGC CCGGGAAAGGCCTGGAGTAC ATGGGGCTCA TCTATCCTGG TGACTCTGAC ACCAAATACAGCCCGTCCTT CCAAGGCCAG GTCACCATCT CAGTCGACAA GTCCGTCAGCACTGCCTACT TGCAATGGAG CAGTCTGAAG CCCTCGGACA GCGCCGTGTATTTTTGTGCG AGACATGACG TGGGATATTG CAGTAGTTCC AACTGCGCAAAGTGGCCTGA ATACTTCCAG CATTGGGGCC AGGGCACCCT GGTCACCGTCTCCTCAGGTG GAGGCGGTTC AGGCGGAGGT GGCTCTGGCG GTGGCGGATCGCAGTCTGTG TTGACGCAGC CGCCCTCAGT GTCTGCGGCC CCAGGACAGAAGGTCACCAT CTCCTGCTCT GGAAGCAGCT CCAACATTGG GAATAATTATGTATCCTGGT ACCAGCAGCT CCCAGGAACA GCCCCCAAAC TCCTCATCTATGGTCACACC AATCGGCCCG CAGGGGTCCC TGACCGATTC TCTGGCTCCAAGTCTGGCAC CTCAGCCTCC CTGGCCATCA GTGGGTTCCG GTCCGAGGATGAGGCTGATT ATTACTGTGC AGCATGGGAT GACAGCCTGA GTGGTTGGGTGTTCGGCGGA GGGACCAAGC TGACCGTCCT AGGTGCTAGC ACCACGACGCCAGCGCCGCG ACCACCAACA CCGGCGCCCA CCATCGCGTC GCAGCCCCTGTCCCTGCGCC CAGAGGCGTG CCGGCCAGCG GCGGGGGGCG CAGTGCACACGAGGGGGCTG GACTTCGCCT GTGATATCTA CATCTGGGCG CCCTTGGCCGGGACTTGTGG GGTCCTTCTC CTGTCACTGG TTATCACCCT TTACTGCAAACGGGGCAGAA AGAAACTCCT GTATATATTC AAACAACCAT TTATGAGACCAGTACAAACT ACTCAAGAGG AAGATGGCTG TAGCTGCCGA TTTCCAGAAGAAGAAGAAGG AGGATGTGAA CTGTAA

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety. While this invention has been disclosed with referenceto specific embodiments, it is apparent that other embodiments andvariations of this invention may be devised by others skilled in the artwithout departing from the true spirit and scope of the invention. Theappended claims are intended to be construed to include all suchembodiments and equivalent variations.

What is claimed is:
 1. A T cell comprising a first chimeric antigenreceptor (CAR) and a second CAR, wherein: (a) the first CAR comprises afirst antigen binding domain that targets a first antigen and the firstCAR comprises SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9; (b) thesecond CAR comprises a second antigen binding domain that targets asecond antigen and the second CAR comprises SEQ ID NO: 3; (c) the firstCAR is not capable of inducing cell activation upon binding to the firstantigen and the first CAR increases resistance to antigen-induced celldeath (AICD); and (d) the T cell is selective for a cancer tissue over anormal tissue thereby reducing “on-target” toxicity, while maintainingpotent anti-cancer activity, tumor localization and in vivo persistencewhen compared to a cell comprising only the first CAR, the second CAR,or a conventional first or second generation CAR comprising the first orthe second antigen-binding domain.
 2. The T cell of claim 1, whereinactivation of the T cell is dependent on the binding of the firstantigen binding domain to the first antigen and the binding of thesecond antigen binding domain to the second antigen.
 3. The T cell ofclaim 1, wherein the T cell exhibits anti-tumor immunity when the firstantigen binding domain binds to the first antigen and the second antigenbinding domain binds to the second antigen.
 4. The T cell of claim 1,wherein the T cell exhibits heightened tumor specificity.
 5. The T cellof claim 1, wherein the T cell targets: (a) mesothelin and folatereceptor alpha on ovarian cancer cells; or (b) mesothelin and HER2 onbreast cancer cells.